VEGF in Development

MOLECULAR BIOLOGY INTELLIGENCE UNIT

VEGF in Development Christiana Ruhrberg, PhD Institute of Ophthalmology University College London London, U K

LANDES BIOSCIENCE AUSTIN, TEXAS

USA

SPRINGER SCIENCE+BUSINESS MEDIA NEW YORK, NEW YORK

USA

VEGF IN DEVELOPMENT Molecular Biology Intelligence Unit Landes Bioscience Springer Science+Business Media, LLC ISBN: 978-0-387-78631-5

Printed on acid-free paper.

Copyright © 2 0 0 8 Landes Bioscience and Springer Science+Business Media, LLC All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher, except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information stors^e and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafi:er developed is forbidden. T h e use in the publication of trade names, trademarks, service marks and similar terms even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the authors, editors and pubUsher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, vnth respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urgpd to carefully review and evaluate the information provided herein. Springer Science+Business Media, LLC, 233 Spring Street, N e w York, N e w York 10013, USA http://www.springer.com Please address all inquiries to the Publishers: Landes Bioscience / Eurekah.com, 1002 West Avenue, 2nd Floor, Austin, Texas 7 8 7 0 1 , USA Phone: 5 1 2 / 637 6050; FAX: 512/ 637 6079 h t t p : //www.landesbioscience.com Printed in the United States of America. 9 8 7 6 5 4 3 2 1

Library of Congress Cataloging-in-Publication Data V E G F in development / [edited by] Christiana Ruhrberg. p. ; cm. — (Molecular biology intelligence unit) Includes bibliographical references and index. ISBN 978-0-387-78631-5 (alk. paper) 1. Neovascularization. 2. Vascular endothelial growth factors. I. Ruhrberg, Christiana. II. Series: Molecular biology intelligence unit (Unnumbered) [DNLM: 1. Vascular Endothelial Growth Factor A—physiology. 2. Growth and Development-physiology. Q U 107 V422 2008] QP106.6.V44 2008 6l2.1'3-dc22

2008009257

About the Editor... CHRISTIANA RUHRBERG, PhD, is a Principal Investigator and Lecturer at University College London, working in the Institute of Ophthalmology. Having conducted her PhD thesis research in the laboratory of Dr. Fiona M. Watt at the Imperial Cancer Research Fund, Dr. Ruhrberg received a PhD in Biochemistry from Imperial College London. She subsequently obtained postdoctoral research experience in Dr. Robb Krumlauf s laboratory at the National Institute of Medical Research in London and in Dr. David Shimas laboratory at the Imperial Cancer Research Fund. Being funded largely by the UK Medical Research Council, her laboratory presently investigates the role of VEGF and neuropilins in vascular biology as well as neuronal and glial development.

CONTENTS 1. The Biology of Vascular Endothelial Cell Growth Factor Isoforms Yin-Shan Ng VEGF Isoform Structure Biochemistry of the Major VEGF Isoforms VEGF Isoforms in Vascular Development VEGF Isoforms in Diseases Novel VEGF Isoforms 2. VEGF Receptor Signalling in Vertebrate Development Joaquim Miguel Vieira, Christiana Ruhrherg and Quenten Schwarz Tyrosine Kinase Receptors for VEGF: FLTl and KDR VEGF Isoform-Specific Receptors: Neuropilins and HSPGs 3. VEGF Gene Regulation Marcus Fruttiger VEGF Gene Regulation through the Hypoxia Response Element Post-Transcriptional Regulation of VEGF VEGF Gene Regulation by Transcription Factors Developmental Control of VEGF Expression 4. Embryonic Vasculogenesis and Hematopoietic Specification Lauren C. Goldiey Melissa K Nix and Karen K Hirschi Endothelial Specification and Vasculogenesis Vascular Remodeling Hemogenic Specification and Hematopoiesis 5. Role of VEGF in Organogenesis JodyJ. Haigh Approaches to Study VEGF during Organogenesis VEGF Signalling in the Developing Cardiovasculature VEGF and the Development of Endoderm-Derived Organs VEGF in Mesoderm-Derived Organs VEGF in Ectoderm-Derived Organs (Skin and Nervous System) 6. VEGF and Endothelial Guidance in Angiogenic Sprouting Holger Gerhardt Mechanics of Angiogenic Sprouting The Mouse Retina Model VEGF Gradients Guide Tip Cell Migration Balancing Tip Cell Migration and Stalk Cell Migration through Extracellular VEGF Gradients Tip Cell Formation following VEGF Stimulation Is Controlled by D114/NOTCH1 Signaling

1 2 4 6 9 10 14 16 20 30 31 32 33 34 40 41 43 AA 52 53 55 59 61 62 68 69 71 72 73 75

7. Vascular and Nonvascular Roles of VEGF in Bone Development Christa Maes and Geert Carmeliet Expression and Regulation of VEGF and VEGF Receptors in Bone Cell Types VEGF Is Required for the Formation of the Primary Ossification Center VEGF Is Required for Metaphyseal Bone Development and Longitudinal Bone Growth VEGF Affects Epiphyseal Cartilage Development and Formation of the Secondary Ossification Center

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8. VEGF in the Nervous System Jeffrey M. Rosenstein, Janette M. Krum and Christiana Ruhrherg VEGF Isoforms and VEGF Receptors in the Nervous System VEGF Controls Brain Angiogenesis Neurotrophic Roles of VEGF during Development and in the Adult VEGF and Neurogenesis VEGF Signalling in Neurovascular Patterning VEGF and Nervous System Repair

91

Index

81 84 85 86

91 92 92 95 97 99 105

EDITOR Christiana Ruhrberg Institute of Ophthalmology University College London London, UK Email: [email protected] Chapters 2 and 8

CONTRIBUTORS Note: Email addresses are provided for the corresponding authors of each chapter. Geert Carmeliet Laboratory for Experimental Medicine and Endocrinology K. U. Leuven Leuven, Belgium Chapter 7 Marcus Fruttiger Institute of Ophthalmology University College London London, UK Email: [email protected] Chapter 3 Holger Gerhardt Vascular Biology Laboratory Cancer Research UK—London Research Institute Lincoln's Inn Fields Laboratories London, UK Email: [email protected] Chapter 6 Lauren C. Goldie Department of Pediatrics Children's Nutrition Research Center and Center for Cell and Gene Therapy Baylor College of Medicine Houston, Texas, USA Chapter 4

JodyJ. Haigh Vascular Cell Biology Unit Department for Molecular Biomedical Research Flanders Interuniversity Institute for Biotechnology/Ghent University Ghent, Belgium Email: [email protected] Chapter 5 Karen K Hirschi Department of Pediatrics Children's Nutrition Research Center and Department of Molecular and Cellular Biology Center for Cell and Gene Therapy Baylor College of Medicine Houston, Texas, USA Email: [email protected] Chapter 4 Janette M. Krum Department of Anatomy and Cell Biology The George Washington University Medical Center Washington, DC, USA Chapter 8

Christa Maes Laboratory for Experimental Medicine and Endocrinology K. U. Leuven Leuven, Belgium Email: [email protected] Chapter 7

Jeffrey M. Rosenstein Department of Anatomy and Cell Biology The George Washington University Medical Center Washington, DC, USA Chapter 8

Yin-Shan Ng (OSI) Eyetech Lexington, Massachusetts, USA Email: [email protected] Chapter 1

Quenten Schwarz Institute of Ophthalmology University College London London, UK Email: [email protected] Chapter 2

Melissa K. Nix Department of Molecular and Cellular Biology Center for Cell and Gene Therapy Baylor College of Medicine Houston, Texas, USA Chapter 4

Joaquim Miguel Vieira Institute of Ophthalmology University College London London, UK Chapter 2

PREFACE

T

his book is devoted to vascular endothelial growth factor A (VEGF or VEGFA), a secreted signalling protein of great significance for development and disease in vertebrates. VEGFA controls the proliferation, migration, specialisation and survival of vascular endothelial cells, and it is therefore essential for the establishment of a functional blood vessel circuit. In addition, VEGFA is emerging as a versatile patterning factor for several non-endothelial cell types in vertebrates. Thus, it plays a central role during organ development at multiple levels, including blood vessel growth, vessel-mediated organ induction and tissue differentiation. The discovery of non-endothelial roles for VEGFA initially came as a surprise. However, the recent indentification of a VEGF homolog in the fruit fly Drosophila^' ^ advocated the idea of an ancestral VEGF function in a non-endothelial cell type, as this organism lacks a vascular network. Most likely, VEGF's original role was to serve as a chemoattractant for various cell types, as VEGFA guides migrating haemocytes and border cells in Drosophila, ^'^ Because Drosophila VEGFA bears homology not only to VEGFA but also to the other V E G F superfamily members, i n c l u d i n g the platelet-derived growth factors (PDGFs) and placental growth factor (PGF), it was named VEGF/PDGF-related factor 1 (VPFl). It is now thought that the invertebrate VPFl gene gave rise to the four vertebrate VEGF and P D G F genes during two rounds of genome duplication that were accompanied by functional mutation of the different VEGF gene copies.^ These genome duplications also multiplied the gene for the VPFl receptor, a tyrosine kinase named VPRl.^ The VPRl copies then evolved into the different vertebrate VEGF and PDGF receptors as well as several other receptor tyrosine kinases in the blood cell lineage.^'^ In this fashion, several different ligand/receptor pairs became available to support the evolution of vertebrates with their increasingly complex and sophisticated organ systems. Most notably, the acquisition of a vascular circuit promoted intrauterine embryogenesis and provided the basis for increased organ and hence body size. Two members of the VEGF superfamily in particular were recruited to support blood vessel formation, VEGFA to promote the growth of the inner, endothelial cell lining of blood vessels, and PDGFB to promote the acquisition of a supportive smooth muscle cell coat; in adults, PGF synergises with VEGF and P D G F to promote vascular repair.^ Other VEGF homologs evolved to allow the elaboration of a lymphatic circuit (VEGFC/D)^ or to guide the distribution of glial cell types in the ever-expanding central nervous system (PDGFA).^ However, the boundaries that were initially drawn to help define the function of different VEGF superfamily members are becoming increasingly blurred; for example, VEGFC was recently reported to guide glial cells in the optic nerve,^ whilst VEGFA was found to guide the migration of facial branchiomotor neurons in the brainstem.^

In writing this book, scientists investigating different aspects of VEGF signalling have come together to highlight the central importance of this growth factor for vertebrate development. From their reviews of our present knowledge, it becomes clear that VEGF s versatility as a patterning molecule is linked to its expression as several splice forms that interact with a number of differentially expressed signalling receptors. Moreover, by highlighting the present gaps in our knowledge, the authors of this book set direction to future research. It is therefore hoped that this book will provide valuable insights to those already studying VEGF as well as to those members of the scientific and medical communities that seek to understand vertebrate development and the origins of disease without specific prior knowledge of the field. Christiana Ruhrbergy PhD References 1. Duchek P, Somogjd K, Jekely G et al. Guidance of cell migration by the Drosophila PDGF/ VEGF receptor. Cell 2001; 107(l):17-26. 2. Heino TI, Karpanen T, Wahlsuom G, et al. The Drosophila VEGF receptor homolog is expressed in hemocytes. Mech Dev 2001; 109(l):69-77. 3. Grassot J, Gouy M, Perriere G et al. Origin and molecular evolution of receptor tyrosine kinases with immunoglobuHn-Hke domains. Mol Biol Evol 2006; 23(6): 1232-1241. 4. Carmeliet P. Angiogenesis in health and disease. Nat Med 2003; 9(6):653-660. 5. Tammela T, Petrova TV, Alitalo K. Molecular lymphangiogenesis: new players. Trends Cell Biol 2005; 15(8):434-44l. 6. Fruttiger M, Karlsson L, Hall AC et al. Defective oligodendrocyte development and severe hypomyelination in PDGF-A knockout mice. Development 1999; 126(3):457-467. 7. Le Bras B, Barallobre MJ, Homman-Ludiye J et al. VEGF-C is a trophic factor for neural progenitors in the vertebrate embryonic brain. Nat Neurosci 2006; 9(3):340-348. 8. Schwarz Q, Gu C, Fujisawa H et al. Vascular endothelial growth factor controls neuronal migration and cooperates with Sema3A to pattern distinct compartments of the facial nerve. Genes Dev 2004; 18(22):2822-2834.

Acknowledgments I wish to thank all authors for sacrificing their valuable time to contribute chapters to this book.

CHAPTER 1

The Biology of Vascular Endothelial Cell Growth Factor Isoforms Yin-Shan Ng* Abstract

T

he field of angiogenesis research was literally transformed overnight by the discovery of vascular endothelial growth factor (VEGF). Researchers quickly embraced VEGF in their different areas of vascular and angiogenesis research, and in the last two decades have discovered much about VEGF biology. It is now clear that VEGF is actually a collection of different isoforms. Through differential pre-mRNA splicing and protein processing, one VEGF gene gives rise to several different protein isoforms, which together orchestrate the complex processes of angiogenesis, vessel growth and adult vascular functions. The VEGF isoforms differ biochemically, and genetic experiments in mice have proven that the isoforms have different functions. Furthermore, certain VEGF isoforms associate with and likely play differential roles in various pathologic states. With better understanding of VEGF isoform biology, new insights into the complex mechanisms ofVEGF-mediated vessel growth can be gained. In addition, findings about the specific VEGF isoform functions have important implications for VEGF-mediated therapeutic angiogenesis as well as anti-angiogenic therapy targeting VEGF.

Key Messages • VEGF is a collection of different isoforms • VEGF isoforms have different biochemical properties • VEGF isoforms have overlapping and distinct functions during development • VEGF is multifunctional for endothelial cells and also acts on other cell types • Expression of a specific VEGF isoform is associated with pathological conditions

Introduction Angiogenesis plays a critical role in the progression of many pathologies, including cancer. The search for angiogenic factors in the last decade has been largely driven by the hope that identification of such factors will lead to new treatments for these pathologies. Anti-angiogenic therapy is particularly promising for the treatment of cancer; the strategy of blocking tumor angiogenesis seems to offer the best approach yet for treating tumors resistant to conventional therapies.^ The list of putative angiogenesis factors grows continuously, but VEGF, one of the first angiogenesis factors identified, is widely believed to be the most important regulator of both normal and pathological angiogenesis. VEGF was first purified from tumor cells by Harold Dvorak and coworkers at the Beth Israel Hospital in Boston, USA. This factor was isolated based on its ability to enhance vascular *Yin-Shan Ng, (OSI) Eyetech, 35 Hartwell Ave, Lexington, Massachusetts 02421, USA. Email: [email protected]

VEGF in Development^ edited by Christiana Ruhrberg. ©2008 Landes Bioscience and Springer Science+Business Media.

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VEGF in Development

permeability, and was therefore named "vascular permeability factor" or VPF. Napoleon Ferrara at Genentech subsequendy purified, and later cloned, a factor from medium conditioned by bovine pituitary foUiculostellate cells that induced proliferation of vascular endothelial cells (EC). This substance was named VEGF. ^ When cDNAs from both the Dvorak and the Ferrara studies were sequenced, VEGF and VPF were found to be the same molecule. It is now clear that VEGF elicits an array of EC responses in vitro, including stimulation of proliferation and migration, and induction of proinflammatory gene expression. VEGF has been shown to guide blood cell and EC precursor migration in Drosophila? Xenopus, and likely in Danio rerio, it is possible that in mammals the ancestral role of VEGF is to direct EC precursor, or angioblast, migration.^ The findings in the Drosophilia and in the lower vertebrates suggest that at least the chemotaxic function of VEGF signaling is conserved during evolution, and the EC mitogenic and vascular permeability functions ofVEGF may have evolved in more complex animals to modulate their vascular system. Certain in vivo characteristics of VEGF further illustrate its role as the primary angiogenic factor. VEGF is a secreted angiogenic factor, and thus can act in a paracrine fashion. Expression patterns of VEGF and its endothelial-selective receptors correlate both temporally and spatially with areas of vascular growth in developing embryos and during the female reproductive cycle. '^'^^ Most importandy, embryos lacking either VEGF or its receptors fail to develop functional vessels.^ ' Interestingly, heterozygous VEGF gene inactivation also results in severe disruption of vessel development and embryonic lethality, suggesting that correct dosage of VEGF is critical for normal vascular development. Indeed, further genetic experiments using a hypermorphic VEGF allele confirmed the tight dosage requirement of VEGF in cardiovascular development.^^ Lasdy, antagonists of VEGF or its receptors can effectively block both normal and pathological angiogenesis in animals. "^^ In addition to its role in developmental angiogenesis, VEGF also modulates adult physiological angiogenesis and vessel function in numerous pathologies. In the adult, VEGF participates in regulation of the female reproductive cycle, wound healing, inflammation, vascular permeability, vascular tone, and hematopoiesis.^^ VEGF function also contributes to pathological angiogenesis in disorders such as cancer, rheumatoid arthritis, diabetic retinopathy and the neovascular form of macular degeneration. '^^''^^ VEGF, also called VEGFA, belongs to the cystine-knot superfamily of growth factors that are characterized by the presence of eight conserved cysteine residues.^'^' ^ In addition to VEGFA, this superfamily of hormones and growth factors includes VEGFB, VEGFC, VEGFD, VEGFE encoded by the various o^/Viruses, and placental growth factor (PlGFl and P1GF2 isoforms).^*^ The VEGF family members are active as secreted, glycosylated homodimers.^^ They are closely related to and likely have a common ancestor with the platelet derived growth factor (PDGF) family of growth factors, which includes PDGFB, PDGFC, and PDGFD.^^ VEGF exerts its effects on EC dirough binding to the EC-selective high affinity receptors FLTl (VEGFRl), BCDR (VEGFR2) and neuropilins (NRPl and NRP2).^'^^ VEGF is highly conserved across different vertebrate species, from mammals to fish, at both the polypeptide and genomic structure levels. VEGF protein from one species is therefore often functional toward EC from another species.

VEGF Isoform Structure The VEGF gene is comprised of several exons separated by introns in all species characterized—from human to mouse, fish and frog.'^^''^^ As a result of differential pre-mRNA splicing, a single VEGF gene gives rise to many different VEGF isoforms.'^^ Because the roles of the different VEGF isoforms in nonmammalian species have not been well characterized, and most studies using specific VEGF isoforms have utilized either mouse or human models, this chapter will focus on the human and murine VEGF isoforms. The gene encoding VEGF is located on the short arm of chromosome 6 (6p21.1) in humans and on chromosome 17 (24.20 cM) in the mouse.^^ Both the human and the murine VEGF genes are comprised of eight exons, separated by seven introns (Fig. 1). Although in

The Biology of Vascular Endothelial Cell Growth Factor Isoforms

3

theory any combination of the eight exons is possible, all differentially spliced variants of VEGF isoforms discovered to date contain the first 5 exons (1 to 5) plus different combinations of exons 6 to 8. As a result, all VEGF isoforms contain signal peptides (first 28 residues) and are thus secreted polypeptides, and they can all potentially form homodimers, because the dimerization domain is located in exons 2 to 5 (residues Cys51 and Cys61). Because three residues tially spliced region of the VEGF pre-mRNA is Umited to exons 6 to 8, the number of potential possible combinations of these exons is further increased by the fact that exons 6 and 7 have alternative internal splice donor and acceptor sites that further divide them into two different portions, referred to as 6a and 6b, 7a and 7b, respectively.^'^''^^

Figure 1. Genomic organization of the human VEGF gene and its alternative splice variants. By differential pre-mRNA splicing of exons 6 and 7, the human veg/gene gives rise to various isoforms (the most common 5 isoforms are shown). The different exons (ex) and domain sizes (a.a., amino acid residue) are shown below the gene. The 5'- and 3'-untranslated regions (UTR) are indicated by hatched boxes. Protease cleavage sites (arrowheads) and receptor binding sites (arrows) are indicated on the VEGF206 isoform.

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VEGF in Development

Despite this variety of potential diflPerential splicing combinations, it appears that there are only three major VEGF isoforms produced in all vertebrates, and these differ by the presence or absence of peptides encoded by exons 6 (24 amino acids) and 7 (44 amino acids). The major human VEGF isoforms are VEGF 121, VEGF 165 and VEGF 189, with the numbers indicating the number of amino acids in the mature polypeptides (Fig. 1). The major murine VEGF isoforms are VEGF 120, VEGF 164 and VEGF 188,^^each containing one less amino acid than die human ordiologue. Invivo, VEGF 121(120), VEGF 165(164) and VEGF 189(188) isoforms are produced by certain cells in a tissue-specific pattern with the clear exception of most vascular endothelial cells,*^^ and many of the less-abundant VEGF isoforms are associated with specialized cell types or tumor cells (see below).

Biochemistry of the Major VEGF Isoforms The different VEGF isoforms have distinct biochemical properties (Fig. 2). As VEGF 120(121) does not bind heparan sulfate, it is readily diffusible. VEGF 164(165) has moderate affinity for heparan sulfate; it is partially sequestered on the cell surface and in the extracellular matrix (ECM),^® likely due to heparan sulfate proteoglycans (HSPGs) binding. The HSPGs-binding aaivity of VEGF 164(165) is conferred by the 15 basic residues within the peptide encoded by exon 7, which is defined as the heparin-binding domain of VEGF.^^ VEGF 188(189) has high affinity for heparan sulfate due to the presence of the additional basic residues and a strongly heparin-binding domain encoded by exon 6. As a result, VEGF 188 (189) is mosdy associated with the cell-surface and ECM.^^ (In this chapter, the nomenclature for the human and murine VEGF isoforms will be used interchangeably). The VEGF isoforms localized or sequestered at the cell surface or ECM constitute a reservoir of angiogenic growth factors that can be mobilized by various enzymes. For example, heparinase and matrix metalloproteinases (MMPs) can release matrix-bound VEGF isoforms from HSPGs,^^'^^ whereas plasmin can cleave the heparin-binding domain of the matrix-bound

Figure 2. Distinct biochemistry of the different VEGF isoforms. Schematic representation of the differential extracellular localization of the three murine VEGF isoforms based on their different affinities for heparin sulfate.

The Biology of Vascular Endothelial Cell Growth Factor Isoforms

5

VEGF isoforms, producing a truncated, but biologically active, VEGFl 10 (VEGF109 in the mouse).^^ This enzyme-mediated VEGF release represents a fast and effective way to mobilize VEGF and increase its effective concentration in different local environments. Therefore one of the functions for the different VEGF isoforms is to modulate the availability of VEGF by being sequestering on the cell surface or in the ECM. Although the details of this protease-mediated VEGF release pathway have not been elucidated in vivo, MMP9 plays a major role in VEGF-mediated tumor angiogenesis in the RipTag pancreatic tumor modeP and dturing bone development,^ suggesting that protease-mediated VEGF release does indeed play a prominent role in the VEGF pathway. In addition to releasing matrix-sequestered VEGF, it has been reported that a nMMP with anti-angiogenic properties, ADAMTSl, directly binds to VEGF and dampens downstream VEGF signaling. More recently, it has been reported that many different MMPs can cleave the matrix-bound VEGF isoforms intramolecularly, creating two fragments: a soluble and biologically active N-terminal fragment that can bind to the VEGF receptors, and a shorter matrix-binding C-terminal fragment that has no apparent biological activity. ^^ Interestingly, the authors also reported distinct angiogenic outcomes produced by the matrix-bound VEGF and the MMP-processed soluble N-terminal VEGF fragment, providing further proof that matrix association can modulate the function of the different VEGF isoforms. These findings clearly suggest that interactions of various MMPs and possibly other proteases with VEGF occur in vivo, and that these interactions can direcdy modulate both the availability and the activity of VEGF. Not only do the VEGF isoforms display differences in localization and availability, they also have different affinities for their high affinity receptors, FLTl and KDR, expressed on the EC surface. Binding of FLTl by both VEGF121 and VEGF 165 is inhibited by heparin. Binding of KDR by VEGF 165 is enhanced by low concentrations of heparin, but is inhibited by high concentrations of heparin.^^ Binding of KDR by VEGF 121 is not affected by heparin. Besides differential binding affinities for FLTl and KDR, the neuropilin family of cell-surface receptors exhibits differential specificity for VEGF isoforms. Because NRPl binds to the peptide encoded by exon 7, VEGF165 and probably VEGF189 bind to NRPl, but VEGF121 does not. Although binding by VEGF 165 may not induce NRPl signal transduction direcdy, due to the lack of a conventional signaling cytoplasmic domain, it was reported that cell surface NRPl can increase die binding affinity of VEGF165 for KDR,^^'^^ but not for VEGF121. This suggests that NRPl functions as a coreceptor in EC cells. Interestingly, it has been reported that VEGF 165, but not VEGF 121, can promote survival of breast carcinoma cells in vitro in a neuropilin-dependent manner, suggesting that NRPl may function as a signaling VEGF 165-specific receptor in nonendothelial cells. Indeed, a chimeric receptor containing the transmembrane and intracellular domain of NRPl and the extracellular domain of epidermal growth factor (EGF) was reported to mediate human umbilical vein EC (HUVEC) migration upon EGF stimulation.^^ These results suggest that the NRPl receptor is capable of transducing signals in certain cell types and in certain contexts. Considering the important role of the NRPl receptor reported for vascular development^^'^ and for guidance of EC tip cells and vessels,^'^ more studies into the exact signaling role of the NRPl will provide further insights into the biology of VEGF isoforms and their roles in modulating angiogenesis. Another neuropilin family member, NRP2, has also been shown to bind VEGF 165 but not VEGF 121.^ Interestingly, NRP2 was reported to function as a receptor for the less-abundant VEGF 145 isoform,^ suggesting that both exon 6 and exon 7 ofVEGF can facilitate the binding to NRP2 by VEGF 145 and VEGF 165, respectively. However, the functional role of this selective interaction between VEGF isoforms and NRP2 in angiogenesis remains to be determined. It is likely that the difference in biochemical properties described above translates into distinct biological activities for the various VEGF isoforms. For example, it has been reported that the VEGF isoforms can have different mitogenic activity for EC in vitro and in vivo. However, others have found that VEGF 120 and VEGF 164 do not differ in their ability to support EC proliferation, but that vessel networks differ in tissues developing in the absence of specific

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VEGF in Development

isoforms and in tumors overexpressing the various VEGF isoforms in vivo. '^^' The differences in localization, availability, receptor-binding affinity and bioactivity likely contribute to distinct roles for the individual VEGF isoforms during vascular development, and imply that the presence of the different VEGF isoforms is critical for normal vasctdar development. Therefore, knowledge of the differential functions of the different isoforms will be crucial for designing an effective VEGF-mediated angiogenesis therapy to promote normal vessels growth in patients suffering from ischemic vascular diseases.

VEGF Isoforms in Vascular Development VEGF mediates angiogenesis both during development and in the adult. While the roles of specific VEGF isoforms in normal adult angiogenesis remain largely unexplored, contributions of VEGF isoforms to developmental angiogenesis have been identified using transgenic mouse models. Experiments that direcdy measured the VEGF isoform mRNA levels in the mouse revealed that the relative levels of the three major VEGF isoforms vary among different adult organs. Differential mRNA levels of the three major VEGF isoforms during murine embryonic development also suggest that expression of the different isoforms is developmentally regulated. Whereas the three major VEGF isoforms are expressed in all embryonic organs examined, the relative levels of each isoform varied from organ to organ, and the isoform composition changed over developmental time in the same organ. For example, adult lung, heart and liver express relatively high levels of VEGF 188 mRNA (52% of the total VEGF message in the lung and 36% in the heart and liver), whereas brain, eye, spleen and kidney express relatively low levels ofVEGF 188 mRNA (6%, 5%, 11% and 17% of the total VEGF mRNA, respectively). During development, in the embryonic day 13.5 lung, VEGF 188 makes up only about 10% of total VEGF mRNA, but at embryonic day 17.5, just before birth, about 50% of the total VEGF mRNA produced in the lung is the VEGF 188 isoform. The levels of VEGF 188 remain high at about 50% of total VEGF mRNA in the adult lung. These findings are consistent with the concept that the VEGF isoforms serve different fimctions during vascular development and angiogenesis in the adult. To directly assess the different functions of the three major VEGF isoforms during development and in the adult, and in an attempt to avoid the early embryonic lethality from the total gene inactivation,^^' VEGF isoform-specific gene targeting approaches were used. Using exon-specific deletion and cDNA replacement (knock-in) strategies, VEGF alleles were created that permitted expression of only one of the three VEGF isoforms: VEGF 120, VEGF 164 or VEGF 188.^^'^^ Mice were then generated in which the normal VEGF gene was replaced with one or two isoform-specific alleles. The isoform-specific mouse models yielded two immediate and significant findings. First, heterozygous mice in which one of the two VEGF alleles could produce only a single VEGF isoform were viable and developed normally (Table 1). Given that single-allele VEGF inactivation, as well as the slight increase in VEGF levels resulting from a hypermorphic mutation, both lead to embryonic lethality, ^^ these observations suggest that the isoform-specific allele likely produced similar levels of VEGF transcript compared to the wild-type allele. Indeed, RNase protection assays confirmed that the total levels of VEGF transcript in the heterozygous and the homozygous VEGF isoform-specific mice were comparable to that of wild-type mice.^^ A second important finding was that although not all VEGF isoform-specific homozygous embryos were viable (Table 1), their phenotypes were much less severe than those of VEGF-null mice. This result is consistent with the concept that the different VEGF isoforms all support EC growth, but have subde differences in fimction, such that one isoform can only partially replace the fixnction of the others during embryonic development and in the adult (Table I). Analysis of die VEGF^^^^^^^ and VEGF ^^^^^^^ homozygous mice, in which bodi VEGF alleles produce only a single VEGF isoform (VEGF 120 and VEGF 188, respectively), has yielded fixrther insight into the roles of the VEGF isoforms in vascular development. Homozygous VEGF ^^^^ mice are not viable and exhibit very distinct vascular defects. Most of the VEGF^^^ ^'^^ embryos die in utero, or soon after birth. All VEGF^'^^^^'^^ embryos exhibit decreased angiogenesis,

The Biology of Vascular Endothelial Cell Growth Factor Isoforms

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Table 1. The phenotypes of VEGF isoform-specific mice Homozygous vegf120/120 Perinatal (90%) or early postnatal (10%) lethality. Extensive vascular hemorrhages in most organs. Decreased vascular branching and impaired postnatal angiogenesis^^'^^'"*^ (Fig. 3).

vegf188/188 Embryonic lethality from embryonic day 10 (90%); 10% reach adulthood, though with decreased body size/weight and subtle vascular defects in the retina'*^ (P. D'Amore and Y. S. Ng, unpublished data). Excessive branching and small caliber of vessels in several embryonic organs."*^ Hyper-fused vessel networks in the yolk sac (Fig. 3). Heterozygous vegf120/+ Viable, normal vegf164/+ Viable, normal vegfl88/+ Viable, normal appearance, but decreased appearance, normal vessel appearance'*^ (P. D'Amore and Y. S. Ng, vascular branching in patterning in all organs unpublished data). examined"*^ (P. D'Amore several embryonic organs26'^9'^3 (pjg 3) and Y. S. Ng, unpublished Vascular phenotype not examined. data; C. Ruhrberg and D. T. Shima, unpublished data). vegfl 64/120 V\ab\e, vegfl88/120 V\ab\e, normal vegf188/164 Viable, Compound heterozygous normal (P. D'Amore and (P. D'Amore and Y. S. Ng, normal (P. D'Amore and Y. S. Ng, unpublished data). unpublished data). Normal Y. S. Ng, unpublished data). The vascular Viability suggests that the vascular development in phenotype has not been combination of VEGF164 several organs."*^'"*^ examined. Viability and VEGF120 isoforms These observations suggests that the is sufficient for normal suggest that the combination of VEGF188 vascular development. combination of VEGF188 and VEGF164 isoforms is and VEGF120 isoforms is sufficient for normal sufficient for normal vascular development. vascular development. vegfl64/164 Viable, normal appearance'*^ (P. D'Amore and Y. S. Ng, unpublished data). No obvious vascular phenotypes in the embryo (C. Ruhrberg and D.T. Shima, unpublished data). Yolk sac vesselsare patterned normally (Fig. 3).

Mice expressing different combinations of VEGF isoforms were produced by crossing heterozygous mice carrying one wild-type and one isoform-specific vegfa\\e\e.

decreased vascular branching and vascular hemorrhage, ^^ suggesting that the vessels in these mice are leaky and lack the normal vascular morphologv found in wild-type mice. Close examination of the vascular beds in organs from the VEGF^^ ^^^ mice of different developmental stages revealed extensive vascular remodeling defects and decreased vascular branching and density (Fig. 3).^^'^^ These results s u r e s t that the freely diffusible VEGF 120 isoform alone can support the initial stages of vascular development, but that VEGF 120 cannot replace the functions of the heparin-binding VEGF isoforms such as VEGF 164 in the fine patterning of the vasculature. There is also a strong correlation between the severity of vascular phenotypes observed in the VEGF embryos and the organ expression patterns of the various isoforms; that is, the organs that express relatively high levels of the heparin-binding VEGF isoforms (VEGF 164 and VEGF 188) exhibited the most severe, abnormal vascular phenotypes. For example, high levels of

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VEGF in Development

Figure 3. Vascular patterning of the VEGF isoform-specific mice. Embryonic day 10.5 yolk sacs showing the different vascular patterns formed in the VEGF120, VEGF164, and VEGF188 isoform-specific mice. In the heterozygous (HET) and homozygous (HOM) VEGF120 yolk sacs, the lack of vessel sprouting results in sparser vascular networks as compared to wildtype (WT), whereas the H O M VEGF188 yolk sac displayed enlarged and sheet-like vessels likely resulting from fusion of vessels due to excessive vascular sprouting and/or branching. Vessels in the VEGF164 yolk sacs are indistinguishable from the WT.

VEGF 188 expression are temporally and spatially associated with the maturation of the lung, and analysis of the lungs from VEGF^^^^^^^ mice revealed extensive microvascular defects and retarded alveolarization. Thus, although the VEGF 120 isoform alone appears to support initial vessel growth in the lung, remodeling or maturation/maintenance of the final vascular bed, including further angiogenesis, is defective in the absence of the heparin-binding VEGF isoforms. Because the VEGF^"^^^^^^ mice produce only freely soluble VEGF, defects in these mice provide information about potential roles of heparin-binding isoforms such as VEGF 164. Analysis of organs in VEGF ^^^^^^ mice that are vascularized by angiogenic sprouting, such as the hindbrain suggested that matrix-bound VEGF 164 is required to establish a normal concentration gradient of VEGF. This concentration gradient, formed when VEGF 164 binds to the cell surface and ECM, can effectively direct the chemotactic activity of the EC by modulating the behavior of filopodia on endothelial vessel tips during vessel sprouting (see Chapter 6 by H. Gerhardt). As the VEGF 120 isoform is freely diffusible, these gradients cannot form properly in VEGF ^^^^^^ mice. These observations establish the importance of localized VEGF in establishing a concentration gradient, and help to explain the unique role of the heparin-binding function of VEGF isoforms in vascular development. Y£Qpi88/i88 jjjj^^ |.QQ^ display abnormal vascular development. About half of the homozygous VEGF^^^ embryos die around embryonic day 10.5, exhibiting developmental delay and extensive vascular defects (D'Amore and Ng unpublished data), and the remaining half develop to term with subtle vascular defects in most organs examined."^^ Because the VEGF 188 isoform is highly localized to ECM and cell surface HSPGs, proteolytic cleavage is required to

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release the biologically active form of soluble N-terminal VEGF fragment from the cell surface and ECM in tissue culture experiments.^^'^^'^^ The fact that some VEGF ^^^^^^^ mice are viable suggests that the VEGF 188 isoform is processed into soluble receptor-binding N-terminal fragment and in ECM-binding C-terminal fragments by proteases in vivo. The VEGF^^^ mouse model supports the idea that post-translational proteolytic processing of VEGF isoforms in vivo could potentially create additional isoform proteins beyond those formed by differential pre-mRNA splicing. However, the functional significance of post-translational modification/processing of VEGF isoforms remains unclear, and more studies will be required to elucidate the biology of this pathway in vascular development and neoangiogenesis.

VEGF Isoforms in Diseases A theory is emerging that particular VEGF isoforms might associate with certain disease states; ^ in this case, specific VEGF isoforms might be considered pathological. Although the mechanism of the disease/pathological VEGF isoform association is unclear, understanding of isoform specificities for diseases might permit development of better and more specific anti-VEGF therapies to treat these conditions. Amongst the better-defined VEGF-mediated pathologies are the proliferative intraocular neovascular syndromes in diseases such as diabetic retinopathy, retinopathy of prematurity (ROP), and the wet form of macular degeneration. In all of these disease states, VEGF not only causes uncontrolled neovascular growth that damages the retina, but also promotes vascular leakage and vitreous hemorrhages that eventually lead to blindness.^^ Recently, VEGF 164 has been identified as the major pathological VEGF isoform in the eye. ^' In experimental murine models of diabetes and ROP, VEGF 164 was more potent than VEGF 120 in inducing both endothelial intercellular adhesion molecule 1 (ICAM-1) expression and chemotaxis of leukocytes, which together lead to increased inflammation of the retinal vessels. Furthermore, VEGF 164 was more potent than VEGF 120 in inducing vascular leakage and blood-retinal barrier breakdown. Interestingly, the vascular inflammation and leakage caused by VEGF in these models were significandy reduced following administration of a pan-VEGF isoform antagonist or a VEGF164-specific, RNA-based aptamer antagonist.^^ These observations suggest that VEGF 164 is the major disease-causing isoform in models of neovascular eye disease, and highlight the importance of understanding the different contributions of specific VEGF isoforms in vascular pathologies. Therefore, anti-angiogenic therapy targeted to individual VEGF isoforms might increase the specificity and potentially the efficacy of the therapy. Another disease that is dependent on angiogenesis is cancer, as solid tumors incorporate new vessels to support and promote tumor growth and metastatic spread. Although not all tumor angiogenesis is VEGF dependent, most tumor types studied to date display upregulation of VEGF mRNA and protein."^ It is therefore likely that VEGF is at least partly responsible for tumor angiogenesis in most cancer types. Interestingly, tumor vessels are mostly unstable, leaky and immature; these characteristics result from the high levels ofVEGF in the tumor. VEGF 164 and VEGF 120 appear to be the most widely expressed VEGF isoforms in tumors. One early study in which murine brain tumors over-expressed one of the three major VEGF isoforms, showed that both VEGF 120 and VEGF 164 promoted rapid growth of vessels that were highly unstable and leaky. In contrast, the VEGF 188 over-expressing brain tumor supported vessels that were slower growing, nonhemorrhagic vessels that were relatively normal in appearance. These results demonstrated that all three VEGF isoforms can stimulate angiogenesis, but that the characteristics of the resulting vessels depend on the specific isoform(s) expressed. Because this tumor study was complicated by the fact that VEGF was also endogenously expressed by the tumor cells, a more refined study was conducted in which tumor cells derived from VEGF-deficient fibroblasts were used to create VEGF isoform-specific tumor cells.^2 jj^ the mouse, these VEGF isoform-specific cells all supported vessel growth in the fibrosarcomas, but the new tumor vessels displayed different vascular characteristics depending on the expressed isoform. The VEGF 164-expressing cells induced tumor growth and vessel density similar

10

VEGF in Developmen t

to those observed for wild-type tumor cells. The VEGF120-speci£ic cells only partially rescued tumor growth, and vascular density in these tumors was reduced. Although the VEGF188-specific cells formed smaller tumors than wild type cells, the vessels in these tumors were present at a higher density and displayed a more highly branched phenotype than observed for the wild type tumor. Furthermore, by mixing the VEGF 120- and VEGF188-specific cells, both the vascular density and tumor growth were comparable to those in the wild type tumor. Taken together, these results suggest that different VEGF isoforms play distinct but cooperative roles during tumor angiogenesis. Although VEGF 164 and VEGF 120 were more potent than VEGF 188 in inducing both tumor growth and angiogenesis, the isoform-specific fibrosarcomas also demonstrated that the VEGF isoforms could act cooperatively to make normal-appearing and functional vessels during tumor vascularization. ^ Specific VEGF isoform expression has also been associated with the progression of a particular tumor type in humans, and may be an indicator for the prognosis of the disease. For example, high levels of VEGF 189 expression were associated with nonsmall-cell lung cancer (NSCLC) and were an indicator of poor prognosis in patients, compared to NSCLC expressing low levels of VEGF 189. High levels of VEGF 165 expression were associated with poor prognosis in osteosarcoma. ^ Although it is not clear whether the predominance of a particular VEGF isoform in a tumor is the cause or a consequence of the disease, such associations might be used as disease markers with potential diagnostic or therapeutic value. ^

Novel VEGF Isoforms With the improved sensitivity of RT-PCR detection techniques and better design of PCR primers based on the VEGF genomic DNA sequence, many novel and low-expression VEGF isoforms have been discovered in diverse tissue types. Relatively low levels of VEGF206 (exons 1-5, 6a, 6b, 7 and 8) and VEGF 145 (exons 1-5, 6a, 8) transcripts have been detected in normal human placental tissues, and VEGF 145 was the major isoform expressed in several carcinoma cell lines derived from the female reproductive system. ^ VEGF 183 (exons 1-5, truncated 6a, 7 and 8) is similar to VEGF 189 but with a shorter exon 6a, and has been reported to have wide tissue distribution.^^ A new heparin-binding VEGF 162 (exon 1-5, 6a, 6b, 8) has recendy been discovered in human ovarian carcinoma cells, and has been reported to have angiogenic activity. VEGF 165b (exon 1-5, 7, distal splice site of exon 8) is very similar to VEGF 165 in protein sequence and size, but inhibits VEGF 165 by competing for KDR binding,^^ and thus may function as an endogenous, competitive inhibitor of all VEGF isoforms for receptor binding. Pre-mRNA splicing is very important in normal development, as it creates protein diversity in complex organisms. This mechanism is also a natural target for various disease processes, including carcinogenesis. Since mice expressing only VEGF 164 are viable, the novel isoforms may be unnecessary for the normal development and survival of adults (Table 1). These novel isoforms may, however, play important roles in disease states. Because upregulation of novel VEGF isoforms has been observed predominately with abnormal tissues, it will be important to determine if these isoforms play a role in disease progression. In vivo animal models will be needed to correlate and confirm expression of novel VEGF isoforms, to exclude potential tissue culture artifacts. Further studies of these isoforms, with their different combinations of C-terminal exons, might help to better elucidate the functions of VEGF in vivo.

Conclusions The central role of VEGF in both normal and pathological angiogenesis has been established, and the complex biology of VEGF, including roles of the different isoforms and receptors, is starting to be elucidated. From the vascular biology point of view, however, much remains unclear regarding the differential fiinctions of the VEGF isoforms. For example, do specific pairings of isoforms with VEGF receptors contribute to the different functions of the VEGF isoforms? Do the isoforms have differential effects on vascular permeability, which is a major problem associated with many vascular pathologies? VEGF was once considered an

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endothelial-specific growth factor that mediating angiogenesis and permeabiUty, but it is now clear that VEGF has additional functions beyond the vasculature that affect cell types including, for example, neural cells, bone-forming cells and immune cells (see Chapters 8 by J. Haigh, J.M. Krum and C. Ruhrberg; Chapter 7 by C. Maes and G. Carmeliet; and Chapter 3 by J.J. Haigh). The roles of the VEGF isoforms in mediating inflammation are poorly understood. Furthermore, the differential effects of the VEGF isoforms on the nervous system, likely via the N R P l / 2 receptors, in development and in diseases remain largely unexplored. With the increasing interest in the use of anti-VEGF therapy to treat various diseases that are associated with abnormal angiogenesis, inflammation, and vessel hyperpermeability, more attention must be paid to understanding the exact roles of VEGF isoforms in normal physiology and vascular pathologies. A better understanding of the isoform functions in building and maintaining normal vessels during development will also allow the design of better pro-angiogenic therapies; specific VEGF isoforms might be particularly beneficial for patients suffering from ischemic diseases.

Acknowledgements The author would like to thank Patricia A. D'Amore for providing Fig. 3 and Anne Goodwin for critical reading of the manuscript.

References 1. Folkman J. Role of angiogenesis in tumor growth and metastasis. Semin Oncol 2002; 29(6 Suppl 16):15-18. 2. Senger DR, Connolly DT, Van de Water L et al. Purification and NH2-terminal amino acid sequence of guinea pig tumor-secreted vascular permeability factor. Cancer Res 1990; 50(6): 1774-1778. 3. Ferrara N, Henzel WJ, Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun 1989; 161(2):851-858. 4. Ferrara N. Vascular endothelial growth factor: Basic science and clinical progress. Endocr Rev 2004; 25(4):581-611. 5. Cho NK, Keyes L, Johnson E et al. Developmental control of blood cell migration by the Drosophila VEGF pathway. Cell 2002; 108(6):865-876. 6. Cleaver O, Krieg PA. VEGF mediates angioblast migration during development of the dorsal aorta in Xenopus. Development 1998; 125(19):3905-3914. 7. Liang D, Chang JR, Chin AJ et al. The role of vascular endothelial growth factor (VEGF) in vasculogenesis, angiogenesis, and hematopoiesis in zebrafish development. Mech Dev 2001; 108(l-2):29-43. 8. Traver D, Zon LI. Walking the walk: Migration and other common themes in blood and vascular development. Cell 2002; 108(6):731-734. 9. Breier G, Albrecht U, Sterrer S et al. Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation. Development 1992; ll4(2):521-532. 10. Millauer B, Wizigmann-Voos S, Schnurch H et al. High affinity VEGF binding and developmental expression surest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell 1993; 72(6):835-846. 11. Fong GH, Rossant J, Gertsenstein M et al. Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endotheHum. Nature 1995; 376(6535):66-70. 12. Shalaby F, Rossant J, Yamaguchi TP et al. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 1995; 376(6535):62-66. 13. Carmeliet P, Ferreira V, Breier G et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996; 380(6573):435-439. 14. Ferrara N, Carver-Moore K, Chen H et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 1996; 380(6573):439-442. 15. Miquerol L, Langille BL, Nagy A. Embryonic development is disrupted by modest increases in vascular endothelial growth factor gene expression. Development 2000; 127(18):394l-3946. 16. Kim KJ, Li B, Winer J et al. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour grow^th in vivo. Nature 1993; 362(6423):84l-844. 17. Millauer B, Shawver LK, Plate KH et al. Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant. Nature 1994; 367(6463):576-579.

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18. Adamis AP, Shima D T , Tolentino MJ et al. Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a n o n h u m a n primate. Arch Ophthalmol 1996; 114(1):66-71. 19. Ferrara N . Role of vascular endothelial growth factor in regulation of physiological angiogenesis. A m J Physiol Cell Physiol 2 0 0 1 ; 280(6):C1358-1366. 20. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. N a t M e d 1995; 1(1):27-31. 2 1 . Adamis AP, Shima D T . T h e role of vascular endothelial growth factor in ocular health and disease. Retina 2005; 25(2):111-118. 22. Holmes D I , Zachary I. T h e vascular endothelial growth factor (VEGF) family: Angiogenic factors in health and disease. Genome Biol 2005; 6(2):209. 2 3 . Robinson CJ, Stringer SE. T h e spUce variants of vascular endothelial growth factor (VEGF) and their receptors. J Cell Sci 2 0 0 1 ; l l 4 ( P t 5):853-865. 24. Soker S, Takashima S, Miao H Q e t al. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 1998; 92(6):735-745. 2 5 . Tischer E, Mitchell R, Hartman T et al. T h e h u m a n gene for vascular endothelial growth factor. M u l t i p l e protein forms are encoded t h r o u g h alternative exon splicing. J Biol C h e m 1 9 9 1 ; 266(18):11947-11954. 26. N g YS, Rohan R, Sunday M E et al. Differential expression of V E G F isoforms in mouse during development and in the aduk. Dev Dyn 2 0 0 1 ; 220(2): 112-121. 27. Miquerol L, Gertsenstein M , Harpal K et al. Multiple developmental roles of V E G F suggested by a L a c Z - t a ^ e d allele. Dev Biol 1999; 212(2):307-322. 2 8 . H o u c k KA, Leung D W , Rowland A M et al. Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms. J Biol C h e m 1992; 267(36) :26031-26037. 29. Bergers G, Brekken R, McMahon G et al. Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol 2000; 2(10):737-744. 30. Engsig M T , Chen QJ, Vu T H et al. Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones. J Cell Biol 2 0 0 0 ; 151(4):879-889. 3 1 . Iruela-Arispe ML, Carpizo D , Luque A. A D A M T S l : A matrix metalloprotease with angioinhibitory properties. A n n N Y Acad Sci 2003; 995:183-190. 32. Lee S, Jilani SM, Nikolova G V et al. Processing of V E G F - A by matrix metalloproteinases regulates bioavailability and vascular patterning in tumors. J Cell Biol 2005; 169(4):681-691. 3 3 . Whitaker G B , Limberg BJ, Rosenbaum JS. Vascular endothelial growth factor receptor-2 and neuropilin-1 form a receptor complex that is responsible for the differential signaling potency of VEGF(165) and VEGF(121). J Biol Chem 2 0 0 1 ; 276(27):25520-25531. 34. Bachelder RE, Crago A, C h u n g J et al. Vascular endothelial growth factor is an autocrine survival factor for neuropilin-expressing breast carcinoma cells. Cancer Res 2 0 0 1 ; 61(15):5736-5740. 3 5 . W a n g L, Zeng H , Wang P et al. Neuropilin-1-mediated vascular permeability factor/vascular endothelial grovnh factor-dependent endothelial cell migration. J Biol C h e m 2003; 278(49):48848-48860. 36. Kawasaki T , Kitsukawa T, Bekku Y et al. A requirement for neuropilin-1 in embryonic vessel formation. Development 1999; 126(21):4895-4902. 37. Gerhardt H , Ruhrberg C, Abramsson A et al. Neuropilin-1 is required for endothelial tip cell guidance in the developing central nervous system. Dev Dyn 2004; 231(3):503-509. 38. Gluzman-Poltorak Z, Cohen T , Herzog Y et al. Neuropilin-2 is a receptor for the vascular endothehal growth factor (VEGF) forms V E G F - 1 4 5 and V E G F - 1 6 5 [corrected]. J Biol C h e m 2000; 275(24):18040-18045. 39. Carmeliet P, N g YS, Nuyens D et al. Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor isoforms VEGF 164 and VEGF 188. Nat M e d 1999; 5(5):495-502. 40. Stalmans I, N g YS, Rohan R et al. Arteriolar and venular patterning in retinas of mice selectively expressing V E G F isoforms. J Clin Invest 2002; 109(3):327-336. 4 1 . C h e n g SY, Nagane M , H u a n g HS et al. Intracerebral tumor-associated hemorrhage caused by overexpression of the vascular endothelial grovnh factor isoforms VEGF 121 and VEGF 165 but not V E G F 1 8 9 . Proc N a d Acad Sci USA 1997; 94(22): 12081-12087. 42. Grunstein J, Masbad JJ, Hickey R et al. Isoforms of vascular endothelial growth factor act in a coordinate fashion T o recruit and expand tumor vasculature. Mol Cell Biol 2000; 20(19):7282-7291. 4 3 . Ruhrberg C , Gerhardt H , Golding M et al. Spatially restricted patterning cues provided by h e p a r i n - b i n d i n g VEGF-A control blood vessel b r a n c h i n g morphogenesis. Genes Dev 2 0 0 2 ; 16(20):2684-2698.

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44. Ng YS, D'Amore PA. Therapeutic angiogenesis for cardiovascular disease. Curr Control Trials Cardiovasc Med 2001; 2(6):278-285. 45. Brinkman BM. Splice variants as cancer biomarkers. Clin Biochem 2004; 37(7):584-594. 46. Ishida S, Usui T, Yamashiro K et al. VEGF164-mediated inflammation is required for pathological, but not physiological, ischemia-induced retinal neovascularization. J Exp Med 2003; 198(3):483-489. 47. Jain RK. Molecular regulation of vessel maturation. Nat Med 2003; 9(6):685-693. 48. Nakamura M, Abe Y, Tokunaga T. Pathological significance of vascular endothelial growth factor A isoform expression in human cancer. Pathol Int 2002; 52(5-6):331-339. 49. Lange T, Guttmann-Raviv N, Baruch L et al. VEGF162, a new heparin-binding vascular endothelial growth factor splice form that is expressed in transformed human cells. J Biol Chem 2003; 278(19):17164-17169. 50. Woolard J, Wang WY, Bevan HS et al. VEGF165b, an inhibitory vascular endothelial growth factor splice variant: Mechanism of action, in vivo effect on angiogenesis and endogenous protein expression. Cancer Res 2004; 64(21):7822-7835.

CHAPTER 2

VEGF Receptor Signalling in Vertebrate Development Joaquim Miguel Vieira, Christiana Ruhrberg and Quenten Schwarz* Abstract

T

he secreted glycoprotein vascular endothelial growth factor A (VEGF or VEGFA) affects many different cell types and modifies a wide spectrum of cellular behaviours in tissue culture models, including proliferation, migration, differentiation and survival. The versatility of VEGF signalling is reflected in the complex composition of its cell surface receptors and their ability to activate a variety of different downstream signalling molecules. A major challenge for VEGF research is to determine which of the specific signalling pathways identified in vitro control development and homeostasis of tissues containing VEGF-responsive cell types in vivo.

Key Messages • • • •

VEGF is expressed in different isoforms VEGF isoforms bind different subsets of cell surface receptors VEGF receptors activate a plethora of downstream signalling pathways VEGF receptors mediate different cellular effects

Introduction Vascular Endothelial Growth Factor A (VEGF or VEGFA) is a critical organiser of vascular development due to its ability to regulate proliferation, migration, specialisation and survival of endothelial cells (reviewed in ref. 1). VEGF also affects many other cell types in tissue culture models. For example, it is mitogenic for lymphocytes, retinal pigment epithelium and Schwann cells. It also stimulates the migration of haematopoietic precursors, monocytes/ macrophages, neurons and vascular smooth muscle cells, and it promotes the survival of developing and mature neurons^^ as well as chondrocytes. ^^'^ Differential splicing of the eight exons comprising the VEGF gene iye^d) gives rise to three main isoforms, termed VEGF 121, VEGF 165 and VEGF189 in humans and VEGF120, VEGF164 and VEGF 188 in mice (see Chapter 1 by Y.-S. Ng). All VEGF isoforms bind to two type III receptor tyrosine kinases, FLTl (/7?w-related tyrosine kinase 1, also denominated VEGFRl) and KDR (kinase insert domain containing receptor, also known as FLKl or VEGFR2) (Fig. lA). In contrast, heparan sulphate proteoglycans (HSPGs) and the nontyrosine kinase receptors neuropilin 1 (NRPl) and neuropilin 2 (NRP2) preferentially bind the VEGF isoforms containing the heparin-binding domains, encoded by exons 6 and 7 (Fig. IB). In addition to the versatility provided by the existence of several different VEGF isoforms and VEGF receptors, VEGF signalling attains further plasticity from the association of VEGF receptors with other transmembrane *Corresponding Author: Quenten Schwarz—Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V9EL, UK. Email: [email protected]

VEGF in Development^ edited by Christiana Ruhrberg. ©2008 Landes Bioscience and Springer Science+Business Media.

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Figure 1. Working models for VEGF receptor signalling. (A-D) Schematic illustration of the different human VEGF receptors and their predicted physiological roles in endothelial cells, blood vessels and macrophages. (A) VEGF tyrosine kinase receptors: All VEGF isoforms(VEGF121, VEGF165, VEGF189) bind to homo- or heterodimers of KDR and FLTl. KDR can form higher order complexes with VE-cadherin or integrins. (B) Isoform-specific VEGF receptors: VEGF165, but not VEGF121, binds receptor complexes containing NRPl and HSPGs, or higher order complexes containing additionally FLTl or KDR. VEGF165 and VEGF145 bind NRP2. The neuropilin CUB domains (al and a2) are shown in blue, the coagulation factor VA^III homology domains (bl and b2) are highlighted red, and the MAM domain is coloured green. (C) FLT1 domain structure: The extracellular region consists of 7 Ig-likefolds (shown as spheres); they bind ligands and mediate receptor dimerisation; the cytoplasmic domain contains two kinase domains (light brown cylinders) interrupted by a kinase insert domain; a juxtamembrane domain is thought to inhibit autophosphorylation. Legend continued on following page.

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Figure 1, continued from previous page. FLT1 contains at least 7 known tyrosine phosphorylation sites (indicated by numbers that correspond to the position in the linear protein sequence); presently, in vivo data are lacking that demonstrate which of these tyrosine residues are essential ipor VEGF signalling in macrophages or other cell types. (D) KDR domain structure: The KDR structure is similar to that of FLT1, but KDR lacks a juxtamembrane inhibitory domain. The 7 known tyrosine phosphorylation sites are numbered according to their position in the linear protein sequence. The phosphorylated tyrosine residues are thought to interact with a collection of different proteins; experimentally confirmed interactions are represented by solid arrows, putative interactions with a dashed line. Interacting proteins that have been disrupted by gene targeting in the mouse are boxed. Abbreviation: BV, blood vessel.

proteins to form higher order signalling complexes (Fig. lA). For example, KDR and FLTl interact with integrins and vascular endothelial cadherin (VE-cadherin). In this chapter, we critically review current knowledge of the different VEGF signalling pathways and their interplay during development to extend a more general recent review on VEGF receptors.

Tyrosine Kinase Receptors for VEGF: FLTl and KDR Structure of FLTl and KDR FLTl and KDR are transmembrane glycoproteins of 180 and 200 kDa, respectively. They are closely related to other type III receptor tyrosine kinases, including FMS, KIT and PDGFR, and contain an extracellular domain composed of seven immunoglobulin (Ig)-like folds, a single transmembrane domain, a regidatory juxtamembrane domain and an intracellidar tyrosine kinase domain (Fig. 1). The intracellular tyrosine kinase domain is interrupted by a kinase insert domain and contains several tyrosine residues that mediate the recruitment of downstream signalling molecules upon phosphorylation (Figs. 1C,D). Both KDR and FLTl bind VEGF with high affinity. Mutation analysis of the extracellidar domains of FLTl and KDR revealed that the second and third Ig-like folds contain the high-affinity ligand-binding domain for VEGF, while the first and fourth Ig-like folds regulate ligand-binding and receptor dimerisation, respectively (Figs. 1C,D).^^-^^ In addition to binding VEGF, FLTl also acts as a receptor for VEGFB and PGF (previously known as PIGF), whilst KDR also binds the VEGF homologs VEGFC and VEGFD and the viral VEGFE.^^ Binding ofVEGF by tyrosine kinase receptors promotes their homophilic or heterophilic interaction to activate the kinase domain. '

Expression Pattern of FLTl and KDR KDR and FLTl are expressed in endothelial cells in most, if not all tissues in mouse and human embryos. The expression level of FLTl in vascular endothelium varies with gestational age. Between embryonic days 8.5 and 14 (E8.5 - El4) in the mouse, the Fltl gene is expressed at high levels in endothelial cells, but expression decreases thereafter.^"^ In newborn mice, Fltl expression increases again, and it continues to be expressed in adults,^^ consistent with the idea that it plays a role in the homeostasis of blood vessels. Fltl gene expression is regulated by hypoxia, and a binding site for hypoxia-inducible factor (HIFIA) has been identified in the Fltl promoter.^^ Thus, Fltl is upregulated in vascular smooth muscle cells experiencing hypoxic stress, perhaps to control vascular remodelling or tone. However, further studies are required to fully understand the physiological significance of the transcriptional regulation of FLTl by hypoxia, and how it may complement the regulation of VEGF by hypoxia (see Chapter 3 by M. Fruttiger). In contrast to Vegfa and Fltl, Kdr has no HIFIA binding sites in its promoter region and is therefore not regulated by hypoxia."^^ Kdr is already expressed in mesodermal progenitors of vascular endothelial cells in the yolk sac at E7 in the mouse, and its expression is often used as a marker for these progenitor cells."^^'"^^ Kdr expression remains high on vascular endothelial cells during development, but it declines towards the end of gestation. Nonendothelial expression of KDR has been reported in neurons, osteoblasts, pancreatic ducts cells, retinal progenitor cells, platelets and megakaryocytes (for

VEGF Receptor Signalling in Vertebrate Development example in refs. 29-32). Due to its expression by adult neurons after brain injury, it has been suggested that KDR has a physiological, possibly neuroprotective fiinction (see Chapter 8 by J. Rosenstein, J. Krum and C. Ruhrberg). Like KDR, FLTl is expressed in endothelial progenitor cells and osteoblasts, but additionally in haematopoietic stem cells, macrophages, osteoclasts, dendritic cells, pericytes, smooth muscle cells and placental trophoblasts.

Functional Requirements for FLTl An essential role for FLTl in development is highlighted by the fact that FLTl-deficient mice die in utero between E8 and E9, most likely due to a failure of endothelial cells to assemble into a vascular circuit. The primary defect underlying this phenotype appears to be an altered cell fate determination among mesenchymal cells, which increases haemangioblast numbers. ^^ The defect has been attributed to VEGF hyperactivity subsequent to the loss of endo-thelial FLT Two different hypotheses have been put forward to explain the negative role of FLTl in developmental angiogenesis. The most widely accepted hypothesis suggests that FLTl functions as a cell surface-bound "decoy receptor" to sequester excess extracellular VEGF. In support of this idea, the FLTl kinase domain is not normally active in endothelial cells, even though FLTl has a ten-fold higher affinity for VEGF compared to KDR; in fact, FLTl activation in endothelial cells has only be achieved by overexpression of recombinant protein. ' Moreover, mice expressing a mutant form of FLTl with an inactive tyrosine kinase domain {Fltl TK-/-) have no discernable defects in blood vessel formation, branching or remodelling, even though these mice show deficiencies in VEGF-induced macrophage migration. Finally, membrane tethering of FLTl is essential for vascular development: 50% of mice expressing solely a soluble form of FLTl, which lacks the transmembrane and tyrosine kinase domains, died between E8.5 and E9.0 with a disorganized vascular network, similar to the fiill knockout. ^ However, whilst 50% of mice expressing only a soluble form of FLTl die, the other 50% of mice making only soluble FLTl survive. A soluble form of FLTl is produced endogenously by alternative splicing (sFLTl), raising the possibility that the soluble isoforfn normally cooperates with the membrane-tethered isoform to control vascular development. For example, it is conceivable that membrane bound FLTl functions as a decoy receptor to limit VEGF availability to KDR, whilst sFLTl sequesters soluble VEGF in the endothelial environment to sharpen VEGF gradients (see Chapter 6 by H. Gerhardt). Even though the FLTl tyrosine kinase domain is dispensable for vascular development, FLTl tyrosine kinase signalling significandy promotes pathological angiogenesis. ' Several different explanations have been put forward to explain this difference in developmental and pathological angiogenesis. Firsdy, FLTl upregulation might aaivate PGF- and VEGF-responsive monocytes, which then release pro-angiogenic factors; in agreement with this idea, FLTl tyrosine kinase signalling mediates chemotactic macrophage migration in response to PGF and VEGF, '^^' ' and PGF promotes macrophage survival during tumour angiogenesis. Alternatively, PGF may occupy FLTl binding sites on endothelial cells, allowing VEGF to bind to KDR rather than FLTl; consistent with this suggestion, PGF potentiates mitogenic VEGF activity in endothelial cells in vitro, and it promotes VEGF-induced vascular permeability in vivo.^ It is also possible that PGF binding to FLTl promotes the transphosphorylation of KDR by FLTl in FLTl/KDR heterodimers to increase VEGF/KDR signaUing.^^ Lasdy, PGF activation of FLTl may stimulate vessel formation and maturation indirectly by acting on nonendothelial cell types, for example smooth muscle cells ' or bone-marrow derived cells that are recruited to sites of neovascularisation."^^'^^'^ It is presently debated whether pro-angiogenic bone-marrow derived cells support tumour angiogenesis by differentiating into endothelial cells or by providing perivascular support cells. The recruited perivascular cells have monocyte/macrophage characteristics, such as expression of the integrin C D l l b and the hematopoietic lineage marker CD45; this observation provides a link to the initial suggestion that PGF supports pathological angiogenesis by acting on cells in the monocyte/macrophage

17

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VEGF in Development

lineage. Importantly, the regulation of FLTl by hypoxia (see above) might promote PGF responsiveness of both endothelial cells and macrophages during pathological angiogenesis.

FLTl'Stimulated Signalling Pathways FLTl contains several potential tyrosine autophosphorylation sites (Fig. IC) (reviewed in ref. 56). Whereas a repressor element in the juxtamembrane domain of FLTl inhibits auto-phosphorylation after VEGF binding,^^ this repression appears to be alleviated by an unknown mechanism in monocytes/macrophages. Biochemical assays suggest that the phosphorylated FLTl can recruit several different proteins containing a SRC homology 2 (SH2)-domain; this domain was first identified in the SRC protein kinase. In endothelial cells, phosphorylated KDR preferentially binds to and activates SRC, whereas phosphorylated FLTl preferentially binds two other protein kinases that are closely related to SRC, namely FYN and YES.^^ Mice lacking any one of the SRC family kinases do not suffer developmental defects, but the combined loss of SRC, FYN and YES results in embryonic lethality at E9.5.^^ Lethality may be due to vascular insufficiency downstream of KDR rather than FLTl signalling in endothelial cells (see below). The physiological role of the different SRC family kinases in VEGF/ PGF mediated macrophage migration has not yet been examined, and the identity of the FLTl and KDR phosphotyrosines involved in SRC kinase recruitment are also still unknown. In addition to SRC kinase recruitment, tyrosine phosphorylation of FLTl promotes recruitment of several other SH2 proteins, including phospholipase C gamma (PLCy), SH2-domain containing tyrosine phosphatase 2 (SHP2), the noncatalytic region of tyrosine kinase adaptor protein 1 (NCKl), the class lA phosphatidylinositol 3-kinase (PI3K) and the cellular homolog of the viral oncogene v-crk (Fig. IC). Phosphorylated Y1213, Y1333, Y794 and Yl 169 all recruit PLCy to activate protein kinase C (PKC). Phosphorylated Yl 213 specifically binds SHP2 and NCKl. Phosphorylated Y1213 also activates PI3K, which then catalyses the production of the second messenger lipid PIP3 (Box 1). Y1333 binds CRK (the cellular homolog of v-crk) and NCK. Proteins that bind to phosphorylated Y1242 and Y1327 have so far remained elusive. Interestingly, VEGF and PGF appear to induce phosphorylation of a different subset of tyrosine residues. ^ For example, PGF, but not VEGF binding to FLTl results in Y1309 phosphorylation and activation of the AKT cell survival pathway (see below).

Box 1. Role of class 1A PI3 kinase in vascular growth. The lipid kinases of the PI3 kinase (PI3K) family produce the intracellular messenger PIP3 (phosphatidyl-inositol-3,4,5-triphosphate); one of the major functions of PIP3 is activation of the serine/threonine kinase AKT to stimulate proliferation and prevent apoptosis. The PI3Ks have been grouped into three classes, with the class I family being further subdivided into lA and IB kinases. The class lA PI3Ks signal downstream of receptor tyrosine kinases. A role for class lA PI3Ks in endothelial cells was initially demonstrated in tissue culture models, but has more recently been studied by genetic alteration of the genes encoding its different subunits. Interpretation of the null mutant phenotypes has, however, been complicated by the fact that ablation of any one of the PI3K subunits deregulates other subunits. For example, ablation of the regulatory subunits p85a, p55a or p50 also reduces expression of the p i 10 catalytic subunits. Conversely, ablation of the pi 10a subunit results in over-expression of the p85 regulatory subunit, which has a dominant negative effect on all class lA PI3K proteins. Perhaps the most resounding evidence so far in support of an essential role for class lA PI3Ks in vascular development comes from the endothelial cell-specific knockout of PTEN (phosphatase and tensin homolog), a lipid phosphatase that reverses PI3K signalling. In this mouse model, loss of PTEN results in an overstimulation of endothelial cell proliferation and migration, causing embryonic death at El 1.5. ^

VEGF Receptor Signalling in Vertebrate Development

19

Understanding the physiological significance of the different FLTl signalling pathways has so far proven difficult. Firsdy, SHP2, PI3K, NCK and PLCy all play roles downstream of a variety of tyrosine kinases, and the analysis of null mutants for these genes therefore cannot identify specific requirements for signalling downstream of FLTl or KDR. Secondly, no appropriate tissue culture model with a relevant readout has been identified to evaluate the physiological importance of the different phosphorylated tyrosine residues in FLTl. It would be particularly interesting to learn more about FLTl signalling pathways in the monocyte/macrophage line^e.

Functional Requirements for KDR Consistent with its expression in the mesodermal progenitors of blood islands in the yolk sac, Kdr is required for endothelial and haematopoietic cell differentiation and therefore vasculogenesis and haematopoiesis; thus, loss of KDR function results in embryonic death between E8.5 and 9.5^^ (see Chapter 4 by L. Goldie, M. Nix and K. Hirschi). As KDR is tyrosine-phosphorylated more efficiendy than FLTl upon VEGF binding in endothelial cells (see above), KDR is thought to be principally responsible for VEGF signalling to stimulate the proliferation, chemotaxis, survival, and differentiation of endothelial cells and to alter their morphology; moreover, KDR signalling is thought to stimulate vessel permeability and vessel dilation.^^'^1-^^ However, owing to the early lethality oi Kdr knockout mice, the requirement for KDR in specific stages of vascular development subsequent to vasculogenesis has not yet been formally demonstrated by knockout technology.

KDR'Stimulated Signalling Pathways KDR functions similarly to most tyrosine kinase receptors: it dimerises and is autophosphorylated on several cytoplasmic tyrosine residues upon ligand binding (Fig. ID). Early experiments using recombinant KDR in bacteria and yeast demonstrated that several tyrosine residues are autophosphorylated upon VEGF binding to recruit SH2-domain containing proteins. The following autophosphorylated tyrosine residues were subsequendy identified in human endothelial cells: in the kinase insert domain, Y951 (corresponding to Y949 in the mouse); in the tyrosine kinase domain, Y1054 and Y1059 (corresponding to Y1053 and Y1057 in the mouse); and in the C-terminal domain, Y1175 and Y1214 (corresponding to Yl 173 and Y1212 in die mouse).^ As ob served in the case of FLTl, BCDR phosphotyrosines are recognised by a number of different SH2-domain containing proteins. For example, SRC kinases have been implicated in signalling pathways downstream of Y951 and Y1175 (Fig. ID), and SRC kinases modulate endothelial proliferation and migration in tissue cidture models^^ and during neoangiogenesis in adults.^ To clarify the relative contribution of the different KDR phosphotyrosines to vascular development, we will discuss the phenotypes of mice that either lack single KDR tyrosine residues or the proteins predicted to bind to them following phosphorylation. Human Y951, Yl 175 and Yl 214 have all been implicated in the control of endothelial cell proliferation or migration in culture models. Y951 is selectively phosphorylated in a subset of endothelial cells during development and binds to the T cell-specific adapter molecule (TSAd), which is thought to act upstream of SRC and PI3K (Fig. ID). Even though TSAd is critical for actin reorganization in cell culture models, it is not essential for mouse development. No other protein has so far been identified that interacts functionally with Y951 in endothelial cells, and it is not known if Y951 is essential for vascular development. Y1214 is embedded in a region of KDR that resembles the consensus binding sequence for the growth factor receptor bound protein 2 (GRB2) and has been implicated in the control of actin reorganisation and cell migration through the activation of CDC42 and the mitogen activated protein kinase (MAPK) cascade^^ (Fig. ID). A mouse model for the tyrosine residues corresponding to human Y1214 has been created by replacing Y1212 with a phenylalanine residue; surprisingly, these mutants have no discernable defects.

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VEGF in Development

A mouse model for the tyrosine residue corresponding to human Y1175 has also been created by replacement of Yl 173 with a phenylalanine residue. This mutation results in embryonic lethality between E8.5 and E9.5 with endothelial and haematopoietic defects, similar to those seen in complete KDR knockout mice/^ The essential Yl 175 residue, located in the KDR C-terminal domain, interacts with a number of SH2 domain-containing proteins that are expressed in endothelial cells, including PLCy and the adaptor proteins SHCA, SHCB (also called SCK) and SHB (Fig. ID). Activation of PLCy leads to the activation of PKC to control endothelial cell proliferation via the MAPK pathway in cultured endothelial cells (Fig. ID). Several different MAPK are essential for embryogenesis, with p38 and ERK5 being required for vascular development; however, it is not clear if the effects on blood vessel growth reflect a requirement in endothelial cells or occur subsequent to defective placentation^^. SHCA KO mice suffer from embryonic lethality due to extensive vascular defects. However, SHCA also interacts with other tyrosine kinase receptors that may be involved in vasculogenesis and may therefore not be a specific downstream effector of KDR. SHCB is expressed in developing blood vessels, but SHCB KO mice have no vascular defects, possibly because it acts redundantly with other SHC family members such as S H C A / ^ SHB controls endothelial cell migration through the focal adhesion kinase FAK in a pathway that involves PI3K activation (Fig. ID). Even though SHB has not yet been knocked out in mice, it is essential for blood vessel growth in an embryonic stem cell model of angiogenesis.^ In addition to promoting the proliferation and migration of endothelial cells, VEGF also promotes their survival. Genetic mouse models suggest that VEGF supports endothelial cell survival in vivo by acting both in a paracrine fashion"^^ and in an autocrine loop.^^ In vitro models have identified several different downstream signalling pathways that are activated by VEGF to promote endothelial survival. Paracrine survival signalling in cultured endothelial cells involves the interaction of KDR with cell adhesion molecules of the integrin family, which control cell survival in response to matrix signals in many cell types including endothelium,^ and the interaction of KDR with VE-cadherin, a component of endothelial cell adherens junctions^^ (Fig. lA). Mice lacking VE-cadherin die at 9.5 dpc due to vascular insufficiency, caused by defective blood vessel remodelling and maturation. These defects may be due to reduced activation of anti-apoptotic protein kinases such as AKTl, a protein that promotes endothelial cell survival in vitro and in vivo.^^ AKTl activation normally occurs downstream ofVE-cadherin and VEGF/KDR in a process diat requires SRC and PI3I^>81'82 (p^g ij)) However, AKTlis not essential for vascular development, possibly because it signals redundandy with closely related AKTl and AKT3 proteins. Alternadvely, or additionally, VE-cadherin/KDR interaction may impact on endothelial cell survival by controlling cell surface retention ofKDR^^^It is not known which intracellular effectors play a role in autocrine VEGF survival signalling, as this pathway does not require VEGF secretion and therefore is likely to bypass KDR/ VE-cadherin complexes on the cell surface.

Negative Regulation of KDR Signalling Whereas much effort has been directed at identifying the forward signalling pathways downstream of KDR, the molecular mechanisms that modulate KDR activity have received less attention. Presumably, KDR activation must be downregulated at some point to terminate signalling. The phosphatases SHPl and SHP2 dephosphorylate the nonessential KDR Y1214 residue,^ '^^ and human cellular protein tyrosine phosphatase A (HCPTPA) inhibits VEGFsignalling in tissue culture models, possibly by dephosphorylating KDR to inhibit MAPK activation.^ Unfortunately, the physiological significance of this pathway is unknown.

VEGF Isoform-Specific Receptors: Neuropilins and HSPGs Identification of the Neuropilins as VEGF Receptors An isoform specific VEGF receptor that binds VEGF 165, but not VEGF 121, was first described in human umbilical vein-derived endothelial cells^^ and subsequendy in several

VEGF Receptor Signalling in Vertebrate Development

21

tumour-derived cell lines that lack the expression of other VEGF receptors. ^^ This novel VEGF receptor was purified and identified as neuropilin 1 (NRPl), a 130-kDa type I transmembrane protein (Fig. IB). NRPl had previously been discovered as an axonal adhesion protein in the developing frog nervous system^^'^^ and as a receptor for secreted guidance molecides of the class 3 semaphorin family.^^'^^ Besides NRPl, the neuropilin family includes NRP2.^^ Even though NRPl and NRP2 share only 44% homology at the amino acid level, each protein is highly conserved amongst different vertebrate species, including frog, chick, mouse and human. NRPl and NRP2 bind a different subset of VEGF isoforms and semaphorins in vitro: whereas NRPl preferentially binds VEGF165 and SEMA3A, N R P l binds bodi VEGF165 and VEGF145 as well as SEMA3E^^"^^

Structure ofNeuropilins NRPl and NRP2 have an identical domain structure.^^'^^ Both contain a large N-terminal extracellular domain of approximately 850 amino acids, a short membrane-spanning domain of approximately 24 amino acid residues and a small cytoplasmic domain of 40 residues. The extracellular domain contains two complement-binding (CUB) domains (termed al and a2), two coagulation factor V/VIII homology domains (termed bl and b2) and a meprin (MAM) domain (Fig. IB). The a- and b-domains are crucial for ligand binding, whilst the MAM domain promotes dimerisation and the interaction with other cell surface receptors. The cytoplasmic domain is short and was originally thought to lack signalling motifs, because its deletion did not impair axonal growth cone collapse in response to SEMA3A.^'^ Instead, neuropilins transduce semaphorin signals in neurons through a signalling coreceptor of the plexin family. ' In analogy, it was inferred that neuropilins recruit a coreceptor such as FLTl and KDR to transmit VEGF signals in endothelial cells. In agreement with this idea, N R P l potentiates the signalling of coexpressed KDR in porcine aortic endothelial cells, which surprisingly lack endogenous KDR expression. ^^^ However, the relationship of NRPl and BCDR is different to that of NRPl and plexins: whereas NRPl is the compulsory ligand binding subunit in the semaphorin receptor, KDR does not require NRPl to bind VEGF. Vice versa, recent evidence suggests that NRPl can also signal independendy of KDR in endothelial cells, suggesting that the cytoplasmic tail may have signalling activity after all (see below).

Functional Requirements for Neuropilins in Vascular Development Neuropilins are expressed by several types of embryonic neurons, and their targeted inactivation in the mouse impairs axon guidance and neuronal migration in response to semaphorins. In addition, loss of NRPl disrupts neuronal migration in response to VEGF. ^ In the vasculature, NRPl is preferentially expressed on arterial and brain microvessel endothelium, whereas NRP2 is present on venous and lymphatic endothelium. ' Consistent with a role for NRPl in vascular growth, over-expression of NRPl in mice deregulates angiogenesis, causing embryonic lethality at El7.5; the mutant embryos exhibit excess capillaries and blood vessels, dilation of blood vessels, severe haemorrhage and malformed hearts. ^^^ Mice lacking NRPl die even earlier, at around El2.5, with impaired neural tube vascularisation, agenesis or transposition of the aortic arches, persistent truncus arteriosus and insufficient development of the yolk sac vasculature.^^^ The physiological role of NRPl during vascular development has also been addressed in zebrafish models. In this organism, knockdown of NRPl impairs angiogenic sprouting from the major axial vessels and therefore formation of the intersomitic vessels.^^^ Others have shown that the knockdown of N R P l in zebrafish disrupts even earlier stages of vascular development, including the formation of the dorsal longitudinal anastomosing vessels and the subintestinal vein.^^ Consistent with its expression pattern, mice lacking NRP2 are deficient in the formation of small lymphatics and capillaries, but they show no other obvious cardiovascular abnormalities.^^^ Noteworthy, loss of both NRPl and NRP2 in mice impairs vascular development more severely than loss of NRPl alone, with death at E8.0 due to impaired yolk sac vascularisation;^ ^'^ these data surest that both proteins can partially compensate for each other during the formation of

22

VEGF in Development

arterio-venous circuits. However, the reason why NRP2 is able to compensate for NRPl during vascular development is presendy unclear. The observation that both proteins are expressed in a reciprocal pattern during the segregation of arterio-venous circuit in the chick^ raises the possibility that venous NRP2 function becomes essential only when arterial NRPl expression is lost. Alternatively, NRP2 may be upregulated in NRPl-deficient vascular endothelial cells to compensate for NRPl. Consistent with this hypothesis, NRP2 is able to enhance KDR signalling in porcine aortic endodielial cells,^^^ which lack NRPl.^^ The requirement for NRPl in vascular growth is generally considered to reflect its essential role in promoting VEGF 165 signalling in endothelial cells. In agreement with this idea, mice lacking NRPl specifically in vascular endothelium show impaired microvessel growth in the brain. However, there are some striking differences in the vascular defects caused by loss of N R P l or loss of its VEGF ligands in the developing trunk and central nervous system, with loss of NRP1 causing a more severe vascular deficiency particularly in the brain. ^^ ' ^^'^ These observations suggest that loss of VEGF isoform signalling through NRPl is not entirely responsible for the vascular deficiency of NRPl null mutants, and that NRPl ligands other than VEGF 165 may contribute to vessel patterning. The finding that SEMA3A inhibits endothelial cell migration in vitro by competing with VEGF 165 for binding to NRPl/KDR complexes made it a candidate modulator of neuropilin-mediated vessel patterning in vivo.^^'^ Yet, class 3 semaphorin-signalling through neuropilins is not required for embryonic vascular development. ^^^'^^^ Therefore, the nature of the hypothetical NRPl ligand that cooperates with VEGF 165 during vascular patterning remains elusive.

VEGF165/NRP1 Signalling In analogy to the compulsory recruitment of plexins to transmit semaphorin signals, NRPl was initially proposed to recruit a coreceptor such as FLTl and KDR to transmit VEGF 165 support of this idea, NRPl does not promote the VEGF165-induced chemotaxis of KDR-negative cultured porcine aortic endothelial cells, but when coexpressed with KDR, it enhances chemotaxis more than KDR alone. ^^^ Two alternative hypotheses have been proposed to explain the beneficial effect of NRPl on KDR signalling: Complexes containing bodi KDR and NRPl may bind VEGF165 widi higher affinity dian KDR or NRPl alone,^^^ or N R P l may promote KDR clustering to promote VEGF 165 signalling.^^^ However, other tissue culture models suggest that NRPl may also function in endothelial cells independently of its ability to enhance VEGF/KDR signalling. Firstly, when the extracellular domain of epidermal growth factor (EGF) receptor was fused to a NRPl fragment comprised of its membrane-spanning and cytoplasmic domain, the chimeric receptor promoted endothelial cell migration in response to EGF.^^^ Secondly, the last three amino acid residues of NRPl (SEA-COOH) bind to the neuropilin-interacting protein NIP (also known as GIPC or svnectin),^^^ and this interaction contributes to vascular development in zebrafish and mice.^ One zebrafish study demonstrated that disruption of trunk vessel development by NRPl knock down could be rescued by delivery of full length human N R P l , but not by human NRPl lacking the NIP-binding SEA motif ^^^ Moreover, ectopic expression of N R P l lacking the SEA motif or knockdown of NIP disrupted vessel growth in this study. ^ ^^ Another zebrafish study found that knockdown of NIP affected vascular development at an even earlier stage by impairing dorsal aorta formation.^ In mice, loss of NIP leads to less severe cardiovascular defects than loss of NRPl.^ NIP null mice are born at the expected Mendelian frequency; moreover, the brain and spinal chord are vascularised normally, even though these tissues are severely affected in NRPl null mutants (J. M. V, C. R. and M. Simmons, unpublished observations). However, NIP-deficient mice show a specific defect in arterial development and adult arteriogenesis, with reduced arterial density and branching in the retina, heart and kidney. ^"^ These observations agree with those of other mouse studies, in which loss of VEGF 165 signalling through NRPl affected arterial patterning in the limb skin^^^ and in the retina.^

VEGF Receptor Signalling in Vertebrate Development In summary, NRP1 is likely to play a dual role in vascular growth by enhancing VEGF 1CA signalling through KDR and by promoting VEGF 164 signalling through its own intracellular domain.

Heparan Sulphate Proteoglycans Heparan sulphate proteoglycans (HSPGs) are abundant and highly conserved components of the cell surface and extracellular matrix. They play an important role in the formation and modulation of gradients of heparin-binding growth factors, morphogens and chemokines. Several reports have implicated HSPGs as modulators of VEGF signalling. Firsdy, VEGF 164 and VEGF 188 bind heparin in vitro with different degrees of affinity, depending on the presence/absence of the so-called heparin-binding domains; heparin-binding ability in vitro is thought to indicate HSPG binding in vivo.^^^ In support of this idea, loss of the heparin-binding VEGF isoforms affects VEGF distribution in the extracellular matrix during angiogenic sprouting in the brain and retina.^ '^^^ Heparin also promotes VEGF 165 binding to its receptors jQ)p^i30,i3i ^ ^ j NRPl.^^*^^^ Moreover, when heparan sulphate is enzymatically removed from endothelial cells, KDR phosphorylation is inhibited. ^^'^ The beneficial effect of heparin or heparan sulphate on VEGF signalling may additionally stem from a direct interaction with the VEGF receptors. Consistent with this suggestion, HSPG expression by perivascular smooth muscle cells transactivates endothelial KDR in an embryonic stem cell model of angiogenesis, and possibly facilitates the cross talk between both cell types during blood vessel formation in vivo.^^^ Finally, NRPl may itself become a proteoglycan by post-transcriptional modification with glycosaminglycan side chains of the heparan sulphate or the chondroitin sidphate type, and this modification may enhance VEGF binding.^

Conclusions and Future Perspectives Initially, VEGF signalling pathways were characterised in tissue culture models of endothelium. More recently, the physiological relevance of the different pathways has been addressed with mouse mutants that carry point mutations in single KDR tyrosine phosphorylation sites or harbour null mutations in proteins that interact with these tyrosines. A more complete understanding of KDR signalling will, however, depend on the creation of further mouse mutants lacking other KDR tyrosine residues implicated in intracellular signalling, as well as the design of a novel strategy to study FLTl tyrosine kinase signalling in vivo. Despite the progress made in identifying intracellular adaptor molecules for KDR and FLTl, we still know very litde about the intracellular trafficking of VEGF and its receptor complexes. For example, in some endothelial culture models the phosphorylation of tyrosine residues Y1054 and Y1059 controls internalisation of the VEGF/BCDR complex into clathrin-coated vesicles and endosomes prior to degradation, ^^^ and KDR may also signal from endosomes to promote endothelial cell proliferation.^^ In other endothelial tissue culture models, VEGF stimulates nuclear translocation of KDR.^^^'^^^ In addition, circumstantial evidence is emerging that autocrine VEGF signalling may be based on intracrine signalling; for example, autocrine VEGF survival signalling in endothelial cells does not require VEGF secretion. Further effort should therefore be directed at establishing the physiological significance of intracellular interactions between VEGF and its receptors during development or disease. Owing to the absolute requirement for VEGF during embryogenesis, many previous studies focussed on elucidating the physiological requirement for VEGF signalling pathways in early vascular development. These studies also benefited from the fact that developmental angiogenesis produces a stereotypic pattern of hierarchical blood vessel networks in a well-defined tissue context. In contrast, adult angiogenesis occurs against a backdrop of environmental fluctuations and is influenced by the dynamic interaction of growing vessels with the immune system. Nevertheless, research into developmental VEGF signalling pathways has impacted on our understanding of neoangiogenesis in the adult, owing to the reactivation of VEGF signalling pathways in physiological processes such as wound healing and pathological conditions such as cancer, diabetic retinopathy and ischemic heart disease. Thus, the potential of novel

23

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VEGF in Development

anti-angiogenic therapies can be evaluated in the perinatal rodent eye before being tested in a disease model, because the rodent retina is vascularised only after birth and the eye is easily accessible for drug delivery (e.g., re£ 140). Finally, it will be necessary to extend the study of VEGF signalling pathways to include other VEGF-responsive cell types, most notably circulating progenitors cells (see Chapter 4 by L. C. Goldie, M. K. Nix and K. K. Hirschi), bone cell types (see Chapter 7 by C. Maes and G. Carmeliet) and neuronal progenitors (see Chapter 8 by J. M. Rosenstein, J. M. Krum and C. Ruhrberg). It will be particularly interesting to elucidate if different VEGF-responsive cell types that grow in close spatiotemporal proximity activate distinct VEGF signalling pathways to coordinate their behaviour. For example, VEGF signalling is likely to play a dual role in blood vessels and bone cell types during bone development (see Chapter 7 by C. Maes and G. Carmeliet), and it supports both blood vessel growth and neuronal growth in the angiogenic niche of neurogenesis (see Chapter 8 by J. Rosenstein, J. Krum and C. Ruhrberg). The identification of cell-type specific components in the VEGF signalling pathway might then provide the basis for the creation of selective tools to balance vascular effects of VEGF such as permeability against effects on nonendothelial cell types in novel pro- and anti-angiogenic therapies.

Acknowledgements We thank Dr Patric Turowski for critical reading of the manuscript. J. M. V. holds a PhD studentship from the Funda9ao para a Ciencia eTecnologia (SFRH/BD/17812/2004). Q. S. is supported by an MRC project grant (G0600993).

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41. Waltenberger J, Claesson-Welsh L, Siegbahn A et al. Different signal transduction properties of KDR and Fltl, two receptors for vascular endothelial growth factor. J Biol Chem 1994; 269(43):26988-26995. 42. Olofsson B, Korpelainen E, Pepper MS et al. Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells. Proc Natl Acad Sci USA 1998; 95(20):11709-11714. 43. Gille H, Kowalski J, Li B et al. Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2): A reassessment using novel receptor-specific vascular endotheUal growth factor mutants. J Biol Chem 2001; 276(5):3222-3230. 44. Hiratsuka S, Minowa O, Kuno J et al. Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice. Proc Natl Acad Sci USA 1998; 95(16):9349-9354. 45. Hiratsuka S, Nakao K, Nakamura K et al. Membrane fixation of vascular endothelial growth factor receptor 1 ligand-binding domain is important for vasculogenesis and angiogpnesis in mice. Mol Cell Biol 2005; 25(l):346-354. 46. Kearney JB, Kappas NC, Ellerstrom C et al. The VEGF receptor flt-1 (VEGFR-1) is a positive modulator of vascular sprout formation and branching morphogenesis. Blood 2004; 103(12):4527-4535. 47. Fliratsuka S, Maru Y, Okada A et al. Involvement of Flt-1 tyrosine kinase (vascular endothelial growth factor receptor-1) in pathological angiogenesis. Cancer Res 2001; 6l(3):1207-1213. 48. Autiero M, Waltenbeiger J, Communi D et al. Role of PlGF in the intra- and intermolecular cross talk between the VEGF receptors Fltl and Flkl. Nat Med 2003; 9(7):936-943. 49. Selvaraj SK, Giri RK, Perelman N et al. Mechanism of monocyte activation and expression of proinflammatory cytochemokines by placenta growth factor. Blood 2003; 102(4):1515-1524. 50. Adini A, Kom^a T, Firoozbakht F et al. Placental growth factor is a survival factor for tumor endothelial cells and macrophages. Cancer Res 2002; 62(10):2749-2752. 51. Park JE, Chen HH, Winer J et al. Placenta grovsrth factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/ KDR. J Biol Chem 1994; 269(4l):25646-25654. 52. Parenti A, Brogelli L, Filippi S et al. Effect of hypoxia and endothelial loss on vascular smooth muscle cell responsiveness to VEGF-A: Role of flt-l/VEGF-receptor-1. Cardiovasc Res 2002; 55(1):201-212. 53. Luttun A, Tjwa M, Moons L et al. Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Fltl. Nat Med 2002; 8(8):831-840. 54. Grunewald M, Avraham I, Dor Y et al. VEGF-induced adult neovascularization: Recruitment, retention, and role of accessory cells. Cell 2006; 124(1):175-189. 55. Lyden D, Hattori K, Dias S et al. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat Med 2001; 7(11):1194-1201. 56. Matsumoto T, Claesson-Welsh L. VEGF receptor signal transduction. Sci STKE 2001; 2001(112):RE21. 57. Gille H, Kowalski J, Yu L et al. A repressor sequence in the juxtamembrane domain of Flt-1 (VEGFR-1) constitutively inhibits vascular endothelial growth factor-dependent phosphatidylinositol 3'-kinase activation and endothelial cell migration. EMBO J 2000; 19(15):4064-4073. 58. Chou MT, Wang J, Fujita DJ. Src kinase becomes preferentially associated with the VEGFR, KDR/Flk-1, following VEGF stimulation of vascular endothelial cells. BMC Biochem 2002; 3:32. 59. Stein PL, Vogel H, Soriano P. Combined deficiencies of Src, Fyn, and Yes tyrosine kinases in mutant mice. Genes Dev 1994; 8(17): 1999-2007. 60. Ito N, Huang K, Claesson-Welsh L. Signal transduction by VEGF receptor-1 wild type and mutant proteins. Cell Signal 2001; 13(ll):849-854. 61. Terman BI, Dougher-Vermazen M, Carrion ME et al. Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor. Biochem Biophys Res Commun 1992; 187(3): 1579-1586. 62. Quinn TP, Peters KG, De Vries C et al. Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium. Proc Natl Acad Sci USA 1993; 90(16):7533-7537. 63. Bernatchez PN, Soker S, Sirois MG. Vascular endotheUal growth factor effect on endothelial cell proliferation, migration, and platelet-activating factor synthesis is Flk-1-dependent. J Biol Chem 1999; 274(43):31047-31054. 64. Takahashi T, Yamaguchi S, Chida K et al. A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-A-dependent activation of PLC-gamma and DNA synthesis in vascular endothelial cells. Embo J 2001; 20(ll):2768-2778.

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65. Werdich XQ, Penn JS. Src, Fyn and Yes play diflPerential roles in VEGF-mediated endothelial cell events. Angiogenesis 2005; 8(4):315-326. (yd. Eliceiri BP, Paul R, Schwartzberg PL et al. Selective requirement for Src kinases during VEGF-induced angiogenesis and vascular permeability. Mol Cell 1999; 4(6):915-924. G7. Rajagopal K, Sommers CL, Decker DC et al. RIBP, a novel Rlk/Txk- and itk-binding adaptor protein that regulates T cell activation. J Exp Med 1999; 190(11): 1657-1668. 68. Lamalice L, Houle F, Jourdan G et al. Phosphorylation of tyrosine 1214 on VEGFR2 is required for VEGF-induced activation of Cdc42 upstream of SAPK2/p38. Oncogene 2004; 23(2):434-445. 69. Holmqvist K, Cross MJ, Rolny C et al. The adaptor protein shb binds to tyrosine 1175 in vascular endothelial growth factor (VEGF) receptor-2 and regulates VEGF-dependent cellular migration. J Biol Chem 2004; 279(21):22267-22275. 70. Sakurai Y, Ohgimoto K, Kataoka Y et al. Essential role of Flk-1 (VEGF receptor 2) tyrosine residue 1173 in vasculogenesis in mice. Proc Natl Acad Sci USA 2005; 102(4):1076-1081. 71. Aouadi M, Binetruy B, Caron L et al. Role of MAPKs in development and differentiation: Lessons from knockout mice. Biochimie 2006; 88(9): 1091-1098. 72. Lai KM, Pawson T. The ShcA phosphotyrosine docking protein sensitizes cardiovascular signaling in the mouse embryo. Genes Dev 2000; 14(9):1132-1145. 73. Sakai R, Henderson JT, O'Bryan JP et al. The mammalian ShcB and ShcC phosphotyrosine docking proteins function in the maturation of sensory and sympathetic neurons. Neuron 2000; 28(3):819-833. 74. Kriz V, Agren N, Lindholm CK et al. The SHB adapter protein is required for normal maturation of mesoderm during in vitro differentiation of embryonic stem cells. J Biol Chem 2006; 281(45):34484-34491. 75. Haigh JJ, Morelli PI, Gerhardt H et al. Cortical and retinal defects caused by dosage-dependent reductions in VEGF-A paracrine signaling. Dev Biol 2003; 262(2):225-241. Id. Lee S, Chen IT", Barber CL et al. Autocrine VEGF signaling is required for vascular homeostasis. Cell 2007; 130(4):691-703. 77. Serini G, Valdembri D, Bussolino F. Integrins and angiogenesis: A sticky business. Exp Cell Res 2006; 312(5):651-658. 78. Carmeliet P, CoUen D. Molecular basis of angiogenesis: Role of VEGF and VE-cadherin. Ann NY Acad Sci 2000; 902:249-262, (discussion 262-244). 79. Carmeliet P, Lampugnani MG, Moons L et al. Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis. Cell 1999; 98(2):147-157. 80. Sun JF, Phung T, Shiojima I et al. Microvascular patterning is controlled by fine-tuning the Akt signal. Proc Natl Acad Sci USA 2005; 102(1):128-133. 81. Gerber HP, McMurtrey A, Kowalski J et al. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J Biol Chem 1998; 273(46):30336-30343. 82. Thakker GD, Hajjar DP, Muller WA et al. The role of phosphatidylinositol 3-kinase in vascular endothelial growth factor signaling. J Biol Chem 1999; 274(15):10002-10007. 83. Lampugnani MG, Orsenigo F, Gagliani MC et al. Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments. J Cell Biol 2006; 174(4):593-604. 84. Guo DQ, Wu LW, Dunbar JD et al. Tumor necrosis factor employs a protein-tyrosine phosphatase to inhibit activation of KDR and vascular endothelial cell growth factor-induced endothelial cell proliferation. J Biol Chem 2000; 275(15):11216-11221. 85. Gallicchio M, Mitola S, Valdembri D et al. Inhibition of vascular endothelial growth factor receptor 2-mediated endothelial cell activation by Axl tyrosine kinase receptor. Blood 2005; 105(5):1970-1976. 86. Huang L, Sankar S, Lin C et al. HCPTPA, a protein tyrosine phosphatase that regulates vascular endothelial growth factor receptor-mediated signal transduction and biological activity. J Biol Chem 1999; 274(53):38183-38188. 87. Gitay-Goren H, Cohen T, Tessler S et al. Selective binding of VEGF121 to one of the three vascular endotheUal growth factor receptors of vascular endothelial cells. J Biol Chem 1996; 271(10):5519-5523. 88. Soker S, Fidder H, Neufeld G et al. Characterization of novel vascular endothelial growth factor (VEGF) receptors on tumor cells that bind VEGF 165 via its exon 7-encoded domain. J Biol Chem 1996; 271(10):576l-5767. 89. Takagi S, Tsuji T, Amagai T et al. Specific cell surface labels in the visual centers of Xenopus laevis tadpole identified using monoclonal antibodies. Dev Biol 1987; 122(1):90-100. 90. Fujisawa H, Ohtsuki T, Takagi S et al. An aberrant retinal pathway and visual centers in Xenopus tadpoles share a common cell surface molecule, A5 antigen. Dev Biol 1989; 135(2):231-240.

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91. He Z, Tessier-Lavigne M. Neuropilin is a receptor for the axonal chemorepellent Semaphorin III. Cell 1997; 90(4):739-751. 92. Kolodkin AL, Levengood DV, Rowe EG et al. Neuropilin is a semaphorin III receptor. Cell 1997; 90(4):753-762. 93. Chen H, Chedotal A, He Z et al. Neuropilin-2, a novel member of the neuropilin family, is a high affinity receptor for the semaphorins Sema E and Sema IV but not Sema III. Neuron 1997; 19(3):547-559. 94. Gluzman-Poltorak Z, Cohen T, Herzog Y et al. Neuropilin-2 is a receptor for the vascular endothelial growth factor (VEGF) forms VEGF-145 and VEGF-165 [corrected]. J Biol Chem 2000; 275(24):18040-18045. 95. Takagi S, Hirata T, Agata K et al. The A5 antigen, a candidate for the neuronal recognition molecule, has homologies to complement components and coagulation factors. Neuron 1991; 7(2):295-307. 96. Lee CC, Kreusch A, McMuUan D et al. Crystal structure of the human neuropilin-1 bl domain. Structure 2003; 11(1):99-108. 97. Nakamura F, Tanaka M, Takahashi T et al. Neuropilin-1 extracellular domains mediate semaphorin D/III-induced growth cone collapse. Neuron 1998; 21(5):1093-1100. 98. Rohm B, Ottemeyer A, Lohrum M et al. Plexin/neuropilin complexes mediate repulsion by the axonal guidance signal semaphorin 3A. Mech Dev 2000; 93(l-2):95-104. 99. Tamagnone L, Artigiani S, Chen H et al. Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates. Cell 1999; 99(l):71-80. 100. Soker S, Takashima S, Miao H Q e t al. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 1998; 92(6):735-745. 101. Kitsukawa T, Shimizu M, Sanbo M et al. Neuropilin-semaphorin III/D-mediated chemorepulsive signals play a crucial role in peripheral nerve projection in mice. Neuron 1997; 19(5):995-1005. 102. Kawasaki T, Bekku Y, Suto F et al. Requirement of neuropilin 1-mediated Sema3A signals in patterning of the sympathetic nervous system. Development 2002; 129(3):671-680. 103. Marin O, Yaron A, Bagri A et al. Sorting of striatal and cortical interneurons regulated by semaphorin-neuropilin interactions. Science 2001; 293(5531):872-875. 104. Giger RJ, Cloutier JF, Sahay A et al. Neuropilin-2 is required in vivo for selective axon guidance responses to secreted semaphorins. Neuron 2000; 25(l):29-4l. 105. Chen H, Bagri A, Zupicich JA et al. Neuropilin-2 regulates the development of selective cranial and sensory nerves and hippocampal mossy fiber projections. Neuron 2000; 25(l):43-56. 106. Herzog Y, Kalcheim C, Kahane N et al. Differential expression of neuropilin-1 and neuropilin-2 in arteries and veins. Mech Dev 2001; 109(1): 115-119. 107. Kitsukawa T, Shimono A, Kawakami A et al. Overexpression of a membrane protein, neuropilin, in chimeric mice causes anomalies in the cardiovascular system, nervous system and limbs. Development 1995; 121(12):4309-4318. 108. Kawasaki T, Kitsukawa T, Bekku Y et al. A requirement for neuropilin-1 in embryonic vessel formation. Development 1999; 126(21):4895-4902. 109. Lee P, Goishi K, Davidson AJ et al. Neuropilin-1 is required for vascular development and is a mediator of VEGF-dependent angiogenesis in zebrafish. Proc Natl Acad Sci USA 2002; 99(16):10470-10475. 110. Wang L, Mukhopadhyay D, Xu X. C terminus of RGS-GAIP-interacting protein conveys neuropilin-1-mediated signaling during angiogenesis. FASEB J 2006; 20(9): 1513-1515. 111. Yuan L, Moyon D, Pardanaud L et al. Abnormal lymphatic vessel development in neuropilin 2 mutant mice. Development 2002; 129(20):4797-4806. 112. Takashima S, Kitakaze M, Asakura M et al. Targeting of both mouse neuropilin-1 and neuropilin-2 genes severely impairs developmental yolk sac and embryonic angiogenesis. Proc Natl Acad Sci USA 2002; 99(6):3657-3662. 113. Favier B, Alam A, Barron P et al. Neuropilin-2 interacts with VEGFR-2 and VEGFR-3 and promotes human endothelial cell survival and migration. Blood 2006; 108(4): 1243-1250. 114. Gu C, Rodriguez ER, Reimert DV et al. Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development. Dev Cell 2003; 5(l):45-57. 115. Ruhrberg C, Gerhardt H, Golding M et al. Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis. Genes Dev 2002; 16(20):2684-2698. 116. Gerhardt H, Ruhrberg C, Abramsson A et al. Neuropilin-1 is required for endothelial tip cell guidance in the developing central nervous system. Dev Dyn 2004; 231(3):503-509.

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117. Miao HQ, Soker S, Feiner L et al. Neuropilin-1 mediates coilapsin-1/semaphorin III inhibition of endothelial cell motility: Functional competition of coUapsin-l and vascular endothelial growth factor-165. J Cell Biol 1999; l46(l):233-242. 118. Vieira JM, Schwarz Q, Ruhrberg C. Selective requirements for NRPl ligands during neurovascular patterning. Development 2007; 134(10):1833-1843. 119. Fuh G, Garcia KG, de Vos AM. The interaction of neuropilin-1 with vascular endothelial growth factor and its receptor flt-1. J Biol Chem 2000; 275(35):26690-26695. 120. Whitaker GB, Limberg BJ, Rosenbaum JS. Vascular endothelial growth factor receptor-2 and neuropilin-1 form a receptor complex that is responsible for the differential signaling potency of VEGF(165) and VEGF(121). J Biol Ghem 2001; 276(27):25520-25531. 121. Soker S, Miao HQ, Nomi M et al. VEGF 165 mediates formation of complexes containing VEGFR-2 and neuropilin-1 that enhance VEGFl65-receptor binding. J Gell Biochem 2002; 85(2):357-368. 122. Wang L, Zeng H, Wang P et al. Neuropilin-1-mediated vascular permeability factor/vascular endothelial growth factor-dependent endothelial cell migration. J Biol Ghem 2003; 278(49):48848-48860. 123. Gai H, Reed RR. Gloning and characterization of neuropilin-1-interacting protein: A PSD-95/Dlg/ ZO-1 domain-containing protein that interacts with the cytoplasmic domain of neuropilin-1. J Neurosci 1999; 19(15):6519-6527. 124. Ghittenden TW, Glaes F, Lanahan AA et al. Selective regulation of arterial branching morphogenesis by synectin. Dev Gell 2006; 10(6):783-795. 125. Mukouyama YS, Gerber HP, Ferrara N et al. Peripheral nerve-derived VEGF promotes arterial differentiation via neuropilin 1-mediated positive feedback. Development 2005; 132(5):941-952. 126. Stalmans I, Ng YS, Rohan R et al. Arteriolar and venular patterning in retinas of mice selectively expressing VEGF isoforms. J GUn Invest 2002; 109(3):327-336. 127. Bernfield M, Kokenyesi R, Kato M et al. Biology of the syndecans: A family of transmembrane heparan sulfate proteoglycans. Annu Rev Gell Biol 1992; 8:365-393. 128. Park JE, Keller GA, Ferrara N. The vascular endothelial growth factor (VEGF) isoforms: Differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF. Mol Biol Gell 1993; 4(12):1317-1326. 129. Gerhardt H, Golding M, Fruttiger M et al. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodiafj Gell Biol 2003; 161(6):1163-1177. 130. Tessler S, Rockwell P, Hicklin D et al. Heparin modulates the interaction of VEGF 165 with soluble and cell associated flk-1 receptors. J Biol Ghem 1994; 269(17):12456-1246l. 131. Terman B, Khandke L, Dougher-Vermazan M et al. VEGF receptor subtypes KDR and FLTl show different sensitivities to heparin and placenta growth factor. Growth Factors 1994; 11(3):187-195. 132. Ashikari-Hada S, Habuchi H, Kariya Y et al. Heparin regulates vascular endothelial growth factor 165-dependent mitogenic activity, tube formation, and its receptor phosphorylation of human endothelial cells: Comparison of the effects of heparin and modified heparins. J Biol Ghem 2005; 280(36):31508-31515. 133. Jakobsson L, Kreuger J, Holmborn K et al. Heparan sulfate in trans potentiates VEGFR-mediated angiogenesis. Dev Gell 2006; 10(5):625-634. 134. Shintani Y, Takashima S, Asano Y et al. Glycosaminoglycan modification of neuropilin-1 modulates VEGFR2 signaling. Embo J 2006; 25(13):3045-3055. 135. Dougher M, Terman BI. Autophosphorylation of KDR in the kinase domain is required for maximal VEGF-stimulated kinase activity and receptor internalization. Oncogene 1999; 18(8): 1619-1627. 136. Feng Y, Venema VJ, Venema RG et al. VEGF induces nuclear translocation of Flk-1/KDR, endothelial nitric oxide synthase, and caveolin-1 in vascular endothelial cells. Biochem Biophys Res Gommun 1999; 256(1): 192-197. 137. Li W, Keller G. VEGF nuclear accumulation correlates with phenotypical changes in endothelial cells. J Gell Sci 2000; 113(Pt 9):1525-1534. 138. Ilan N, Tucker A, Madri JA. Vascular endothelial growth factor expression, beta-catenin tyrosine phosphorylation, and endothelial proliferative behavior: A pathway for transformation? Lab Invest 2003; 83(8):1105-1115. 139. Santos ^Q, Miguel G, Domingues I et al. VEGF and VEGFR-2 (KDR) internalization is required for endothelial recovery during wound healing. Exp Gell Res 2007; 313(8):1561-1574. 140. Pan Q, Ghanthery Y, Liang WG et al. Blocking neuropilin-1 function has an additive effect with anti-VEGF to inhibit tumor growth. Cancer Cell 2007; ll(l):53-67. 141. Hamada K, Sasaki T, Koni PA et al. The PTEN/PI3K pathway governs normal vascular development and tumor angiogenesis. Genes Dev 2005; 19(17):2054-2065.

CHAPTER 3

VEGF Gene Regulation Marcus Fruttiger* Abstract

V

EGF is best known for its angiogenic properties. Not only does it promote the growth of new blood vessels during embryonic development, it is also important in the adult, where it plays a role in maintaining an adequate supply of oxygen and nutrients to most tissues. VEGF gene regulation is controlled by different signalling pathways depending on the context in which it is expressed. Best imderstood is the induction of VEGF expression by hypoxia in neonates and adults, which represents an adaptive response to metabolic stress. In contrast, the mechanisms that control VEGF expression during embryonic development are currently less clear.

Key Messages • • • • •

VEGF is a multifunctional molecule that is regulated by numerous different signalling pathways. VEGF expression is induced by hypoxia. Hypoxia stimulates V^GF transcription by increasing HIF activity. Hypoxia stimulates VEGF translation by increasing mRNA stability. V^GFgene expression is also controlled by hypoxia-independent mechanisms, in particular during embryogenesis.

Introductioii More than half a century ago, Michaelson used an ink perfusion technique to visualize the developing retinal vasculature and noticed that capillary growth near veins was much more vigorous than near arteries. He proposed the existence of a vasoformative molecule termed factor X, which is (a) produced by extra-vascular tissue, (b) distributed in a gradient and (c) antagonized by oxygen. As we now know, these criteria are fulfilled by the vascular endothelial growth factor VEGF, also known as VEGFA. A team lead by Eli Keshet was the first to propose that VEGF could be the long elusive factor X that mediates hypoxia-induced vascular growth.^ This was based on the knowledge that VEGF had already been shown to promote the growth of blood vessels,^ and on the observation that VEGF mRNA dramatically increases under hypoxic conditions in various cell lines. In addition, it was found that VEGF levels were increased in the hypoxic centre of tumours, suggesting that this factor could mediate the growth of new vessels into tumours. This had important clinical implications, because it was known since the early seventies that tumour growth requires sprouting of new vessels from preexisting vessels, a process known as angiogenesis. The discovery of an angiogenic factor in tumours provided for the first time a molecular target for anti-angiogenesis therapy in the fight against cancer, triggering massive research on VEGF. In 1996 two teams simultaneously reported the *Marcus Fruttiger—UCL Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V9EL, UK. Ennail: [email protected]

VEGF in Development^ edited by Christiana Ruhrberg. ©2008 Landes Bioscience and Springer Science+Business Media.

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genetic deletion of VEGF in mice.^'^ Both groups found that the inactivation of just one VEGF dl\&\Q caused early embryonic lethality. This was unexpected and made further experiments technically challenging, as no heterozygous founder mice could be created. It also dramatically illustrated the importance of correct VEGF dosage during embryogenesis. Subsequent research has uncovered a multitude of mechanisms that tightly control VEGF dosage and its biological activity.

VEGF Gene Regulation through the Hypoxia Response Element The most prominent stimulus for VEGF expression is hypoxia. In hypoxic tissue, VEGF is upregulated, and this stimulates blood vessel growth. Increased blood supply then alleviates the hypoxia, turning VEGF expression off again. This simple negative feedback loop ensures that supply and demand of oxygen in tissue are always adequately matched. But how does this process work at a molecular level? A short sequence in the 5' flanking region of the VEGF gene is important for VEGF induction by hypoxia. This sequence element, termed hypoxia response element (HRE), was initially discovered as an enhancer element within the erythropoietin (EPO) gene.^'^^ The element is also present in many other hypoxia inducible genes and is a binding site for the transcription factor hypoxia-inducible factor 1 (HIFl). H I F l is a heterodimer consisting of an alpha and beta subunit, both of which are basic helix-loop-helix PAS domain proteins. ^ The beta subunit is known as the aryl hydrocarbon receptor nuclear translocator (ARNT) and is constitutively expressed, whereas the alpha subunit, H I F l A, is regulated by hypoxia. Although the mRNA encoding HIFl A increases in hypoxic cells, the dominant mechanism of HIFl A regulation is post-translational (Fig. 1). Under normoxic

Figure 1. HIF mediated VEGF transcription. Under normoxic conditions, hypoxia inducible factor (HIF) is hydroxylated by prolyl hydroxylases (PHDs). This modification facilitates binding of the von Hippel Lindau protein (VHL), resulting in ubiquitination and rapid degradation of HIF. At low oxygen concentration, hydroxylation via PHDs becomes less efficient, resulting in HIF accumulation and HIF binding to a hypoxia response element (HRE) in the VEGF promoter.

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conditions, HIFIA is swiftly destroyed via proteasomal degradation. However, when oxygen tension falls, this degradation process becomes less efficient and HIFIA protein rapidly accumulates; a sensible strategy, considering that under stressful conditions it might be easier to stop destroying a protein rather than to start producing new protein. The gene product of the von-Hippel-Lindau (VHL) tumour suppressor gene is critically involved in the degradation of HIFIA. VHL is part of a multi protein complex containing ubiquitin E3 ligase. Binding of VHL to HIFIA results in HIFIA ubiquitination and very rapid proteasomal destruction, making HIFIA one of the most short-lived proteins known. In cells that lack VHL, HIFIA protein levels build up, resulting in increased expression of VEGF and other angiogenic factors. Clinical manifestation of this occurs in von Hippel-Lindau disease, a dominandy inherited familial cancer syndrome predisposing the patient to a variety of malignant and benign tumours. Germline mutations in one allele of VHL do not seem to have any noticeable deleterious effects. However, silencing the second copy of the gene via somatic inactivation or deleterious mutations frequendy leads to haemangioblastomas and renal cell carcinomas. The molecular mechanisms that control binding of VHL to HIFIA are broadly understood and depend on the enzymatic hydroxylation of conserved prolyl residues within the oxygen-dependent degradation domain (ODD) of HIFIA. Deletion of the O D D domain, or mutation of the prolyl residues to alanins, renders HIF resistant to oxygen-induced degradation. The hydroxylation of HIFIA can be carried out by at least three different prolyl hydroxylases, termed PHD I, PHD2 and PHD3 (new names: EGLNl-3). This modification requires the cofactors 2-oxoglutarate, vitamin C, iron and, importantly, molecular oxygen. At low oxygen concentrations, the PHDs become less efficient and HIFIA is no longer hydroxylated, which prevents HIFIA from binding VHL and therefore stops its ubiquitination and degradation; the resulting increased HIF levels ultimately augment VEGF transcription. The pathway shown in Figure 1 is probably the best know mechanism of VEGF regulation to date. However, when the HRE in the VEGF promoter was deleted in mice, they showed an unexpected phenotype.^ Consistent with an important role for HIF mediated VEGF expression during development, half of the mutant mice died embryonically or perinatally. The surviving half of mice lived for up to two years, but gained less weight, were infertile and developped adult-onset motor neuron degeneration resembling amyotrophic lateral sclerosis (ALS). Interestingly, these surviving mice show impaired hypoxia-induced VEGF upregulation in the brain and spinal cord, but not in fibroblasts, confirming the previous finding that the response to hypoxia is differentially regulated in an organ- and even cell type-specific manner. ^^ However, these in vivo findings contrast a number of in vitro experiments, which had convincingly shown that hypoxic VEGF induction critically depends on HIF and the HRE in many different cell types.^ It is possible that HIF influences VEGF gene expression via elements other than the HRE in the VEGF promoter; alternatively, HIF-independent systems may compensate for deficiency in the HRE-mediated hypoxia regulation in some tissues.

Post-Transcriptiotial Regulation of VEGF The mRNAs of house keeping genes such as p-globin or GAPDH have half-lives of over 20 hours, whereas the mRNAs of cytokines or transcriptional activators typically are short lived, with half-lives of 10-30 minutes. Stabilization of normally unstable mRNAs in response to stimuli such as hypoxia, growth factors, hormones or second messengers provides a mechanism to regulate protein levels. Accordingly, Levy et al found that hypoxia increases VEGF mRNA half-life by a factor of 3, resulting in 8-30 times higher levels of VEGF mRNA. In comparison, HIF mediated regulation of VEGF transcription increases VEGF mRNA amounts only 2-3 times. ^ Degradation of mRNA transcripts usually starts with 3'-5' exonucleolytic deadenylation, which removes most or all of the poly(A)-tail. The rest of the mRNA is degraded either by 3'-5' exonucleolytic degradation and/or by removal of the 5* cap, followed by 5'-3' exonucleolytic

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33

degradation. Some mRNAs such as that for insulin-like growth factor are also cleaved by endonucleases, initiating subsequent exonucleolytic degradation. Whilst the proteins that mediate and control this degradation process are only partially understood, a variety of mRNA cis-elements have been identified, which are decisive in determining mRNA stability. In the case of the VEGF mRNA, AU-rich cis-elements (AREs) are important. They are around 50 to 150 nucleotides long with no single conserved consensus motive, but they often contain several copies of the pentamer AUUUA or the nonamer UUAUUUAUU and one or several U-rich stretches. The 3' untranslated region of VEGF mRNA contains a 125bp ARE that is bound by a series of hypoxia-induced proteins, but few have so far been identified. One of the binding partners of the VEGF-ARE is HuR (also known as ELAVLl), a member of the ELAV/Hu family of RNA binding proteins. HuR can stabilize mRNA by displacing or inhibiting factors that cleave or deadenylate ARE-containing transcripts.^^ Interestingly, brain tumours ubiquitously express HuR and display elevated levels of cytokines and angiogenic factors such as VEGF.^^ Poly(A)-binding protein interacting protein 2 (PAIP2) also binds the VEGF-ARE and, via interactions widi HuR, stabilizes die VEGF mRNA^^ A ftirther VEGF-ARE binding protein is zinc finger binding protein 36 (ZFP36L1, also known as TISl IB), but, in contrast to HuR, it has mRNA-destabilizing activity.^^ How hypoxia influences the activity of HuR or other VEGF mRNA-binding proteins remains unclear. An obvious possibility is that the same oxygen sensing machinery that regulates HIF stability feeds into mRNA-degradation mechanisms via interactions with VHL. In fact, there are studies that support such a view. For example, overexpression of VHL in renal carcinoma cells can decrease VEGF mRNA levels via a posttranscriptional mechanism,^^*^^ and rapid turnover of mRNA containing AREs depends on ubiquitination and proteasome activity.^ Furthermore, an association between HuR and VHL was shown in renal cell carcinoma, implicating VHL not only in HIF-mediated VEGF transcription, but also in the cellular signalling events that regulate VEGF mRNA stability.^^ On the other hand, VEGF mRNA can be stabilized by the protein kinase C signalling pathway^ or the stress signalling pathways via p38MAPK and JNK.^^ Whedier diese padiways are used to mediate hypoxia-induced VEGF mRNA stabilization is not known. In addition to mRNA stabilization, the post-transcriptional regulation of VEGF has one more layer of complexity that is based on mRNA translation. Eukaryotic protein synthesis usually depends on binding of the translation initiation complex to the mRNA cap, with ribosomes scanning the 5' untranslated region (UTR) until they encounter the first AUG codon. VEGF possesses an unusually long GC-rich 5' UTR of more than 1000 nucleotides, which can inhibit efficient ribosomal scanning. ^^ An alternative, cap-independent mechanism of translation has been identified in picornavirus, which contains long 5' UTRs without caps, but uses elements termed internal ribosomal entry sites (IRES). It is believed that VEGF mRNA with its cumbersome 5' UTR is usually translated efficiently due to the presence of an IRES element in the 5' UTR. ^^ The cap-independent translation of VEGF mRNA via the IRES element remains efficient under hypoxic stress, whilst overall cap-dependent protein synthesis is reduced by up to 50%,^^ resulting in a relative increase of VEGF translation.

VEGF Gene Regulation by Transcription Factors The signalling pathways that regulate cell metabolism and cell growth are intricately linked with the hypoxia-sensing machinery. This is illustrated by the fact that HIF induces angiogenic genes, but also regulates many aspects of anaerobic metabolism, such as the expression of glucose transporter 1 and most of the glycolytic enzymes. VEGF plays a major role in helping to maintain cellular homeostasis in response to hypoxia, acidosis, UV, lack of nutrients and other stresses by stimulating angiogenesis. Accordingly, VEGF expression is not only controlled by hypoxia, but by a complex regulatory network involving many other signalling pathways, including cytokines and growth factors. ^^ That the regulation of cell metabolism and the hypoxia-sensing machinery are interconnected is also evidenced in the control of VEGF gene

34

VEGF in Development

expression: VEGF is rapidly induced upon serum stimulation in normoxic serum-deprived fibroblasts; this response is mediated by the ERK signalling pathway. ^^ Activation of this pathway stimidates HIF aaivity as well, which in turn also induces VEGF transcription. There are several HIF-independent mechanisms to upregulate VEGF transcription. This is evident from the promoter region of the VEGF gene, which contains binding elements for transcription factors such as specific protein 1 (SPl), signal transducer and activator of transcription 3 (STAT3), activator protein 1 (API) and many others.^^'^ SPl is essential for basal transcription of VEGF. Its phosphorylation following ERK activation leads to VEGF mRNA upregulation independent of hypoxia and HIF.^^ It was also shown that SPl is responsible for the very high levels of constitutively expressed VEGF in many cancer cell lines. Furthermore, tumour suppressors such as p73 or VHL can affect VEGF transcription via SPl.^^'^^ In the case ofVHL, this is based on direct binding to SPl, which inhibits SPl activity. ^^ Thus, loss of VHL not only induces VEGF via HIF activation (see above), but also via de-repression of SPl. STAT3 is able to directly bind the V^G!F promoter to upregulate transcription, and a constitutively active mutant form of STAT3 has been found in various types of tmnours. ' ^ The presence of four candidate API binding sites in the V^GF promoter suggests that API plays a role in VEGF regulation. The API transcription factor is a dimer consisting of basic region-leucine zipper (bZIP) proteins such as Jun and Fos protein family members. It can cooperate with HIF to increase VEGF expression under hypoxic conditions, which involves members of the MAP kinase family. It has also been shown that JUND protects cells from oxidative stress and exerts an anti-angiogenic effect via a HIF-mediated mechanism. ^ Transcription factors that direcdy regulate VEGF transcription, such as HIF, SPl, STAT3 and API, are in turn controlled by a complex network of interacting signalling pathways. Apart from the PHD-VHL pathway, the ERK, JNK and p38 MAP kinase pathways can all modulate VEGF expression; moreover, signalling via PI3K, AKT (also known as RAG) and mTOR (also known as FRAPl) plays a role in T^EGT^gene regulation.^^'^^ Therefore, the simple concept that hypoxia controls HIF to control VEGF levels had to be broadened to include the idea that VEGF transcription and VEGF translation are being influenced by several other signalling pathways (Fig. 2).

Developmental Control of VEGF Expression In all animals, small blood vessels and capillaries are organized stochastically, whereas larger vessels form stereotypical vascular trees. This makes sense, as small vessels grow in response to metabolic demand. On the other hand, large vessels look more or less the same in every person, because they were patterned by "hard wired*' morphogenetic programs operating during embryonic development. VEGF plays a major role in vessel formation both in physiological and pathological angiogenesis in the adult as well as in the developing embryo. We now know that angiogenesis in the adult is to a large extent hypoxia-driven. But is developmental VEGF expression regulated by hypoxia or hypoxia-independent mechanisms? There is evidence supporting both mechanisms of VEGF-stimulated angiogenesis during development. The neonatal retina is a particularly illustrative example of a tissue in which hypoxia plays a major role in VEGF regulation and angiogenesis during development. Aroimd birth in mice and before birth in humans, the retinal vasculature emerges from the optic nerve head and covers the inner retinal surface as a centrifugally spreading vascular plexus. This process is tightly controlled by retinal astrocytes, which also emerge from the optic nerve head and migrate ahead of the growing blood vessel (Fig. 3). These specialized glial cells provide a template for the vascular network and strongly express VEGF. However, as soon as they become covered by the oxygen-carrying vessel network, VEGF is downregulated (Fig. 3). This differential expression of VEGF (low in the centre and high in the periphery of the retina) leads to a VEGF gradient across the retina that is important for the correct outgrowth of the retinal vasculature. Labelling with a hypoxia-probe shows that the peripheral, not yet vascularized region of the retina is indeed hypoxic, whereas the vascularized central portion is not (Fig. 3). This

VEGF Gene Regulation

35

Figure 2. VEGF expression is regulated at the level of mRNA transcription and mRNA stability. VEGF transcription is induced by binding of diverse transcription factors to their recognition elements in the VEGF promoter. VECFmRNA stability is increased by mRNA stabilizing proteins such as HuR (ELAVL1). A complex network of signalling pathv^ays including different kinase pathv^ays and VHL can influence both mRNA transcription and mRNA stability of VEGF.

difference in tissue oxygenation explains the high levels of VEGF mRNA in the periphery and the low levels in the central vascularized portion of the retina. It seems that the hypoxia-driven VEGF gtne^ expression of peripheral retinal astrocytes does not depend on the HRE in the V^67^ promoter, because the retinal vasculature develops normally in mice that lack this sequence in their genome. It is therefore likely that the high levels of VEGF mRNA in peripheral retinal astrocytes are due to increased mRNA stability. Furthermore, retinal astrocytes express much higher levels of VEGF than the underlying neurons, even though both cell populations are likely to experience more or less the same oxygen tension. Retinal astrocytes may therefore be more sensitive to hypoxia than neurons; one could envisage a mechanism involving increased basal activity of the l/EGT^ promoter, caused, for example, by increased SPl levels in retinal astrocytes, but this has not yet been investigated. It is conceivable that in the embryo, similar to the neonatal mouse retina, rapidly expanding tissue outgrow^s its vascular support and becomes hypoxic, inducing VEGF expression. Consistent with this idea, the use of hypoxia probes has revealed that the brain is hypoxic during embryogenesis. ^ In some brain regions, hypoxia-probed labelled areas correspond to areas prominent in HIFIA—and VEGF-immunoreactivity. ^ However, it is unlikely that a hypoxia-controUed mechanism is sufficient to pattern the entire embryonic vasculature. For example, VEGF plays a crucial role very early during development to promote the propagation of the endothelial precursor lineage and in the formation of the first major vessels, the dorsal aorta and the cardinal veins. Yet, these processes occur at a stage when the embryo is still small enough for oxygenation to occur via diffusion from the vascularised yolk sac. In these situations, VEGF is expressed in stereotypical and discrete areas to stimulate reproducible vascular patterning events. These expression patterns are likely "hard-wired", i.e., controlled by hypoxia-independent pathways. Consistent with this notion, sonic hedgehog is secreted from the notochord in zebrafish to induce VEGF expression in adjacent somites.

36

VEGF in Development

Figure 3. VEGF expression during retinal vascularisation. Astrocytes and blood vessels invade the newborn retina from the optic nerve (A). Retinal astrocytes serve as a template and VEGF source for the growing retinal vasculature (B). Astrocytes in the peripheral, not yet vascularized part of the retina experience hypoxia (C) and express VEGF dX higher levels than astrocytes in the vascularized portion of the retina (D). The resulting VEGF gradient stimulates vessel growth towards the periphery. which in turn leads to the formation of the dorsal aorta. Moreover, during brainstem development, VEGF is expressed in stereotypical longitudinal stripes reminiscent of neurogenesis pattern well after the brain has assembled a vascular network to counteract the emergence of

VEGF Gene Regulation

37

hypoxia; these expression patterns Ukely reflect a role in the development of neurons (and glia), rather than a role in blood vessel growth.^^ The signalling pathways that control VEGF expression during vasculogenesis are only beginning to be understood (see Chapter by L.C5. Goldie, M.K. Nix and K.K. Hirschi), and the pathways controlling VEGF expression during neuronal development are not known at all.

Conclusions VEGF is a versatile molecule with different functions in different settings. It acts as a survival factor, mitogen and guidance molecule for endothelial cells. It regulates angiogenesis in response to metabolic needs in the adult, but also participates in the formation of the vascular tree during embryogenesis. In addition, VEGF can regulate vessel permeability and plays a role in neuronal development and neuroprotection. ^^'^ With such a varied array of biological fixnctions it is not surprising that the regulation of VEGF gene expression is diverse. During development, hypoxia-independent pathways likely control VEGF expression to induce the formation of the stereotypically patterned elements of the vascular tree, whereas hypoxia-induced VEGF expression can fine tune the shape and density of capillary networks. Importandy, VEGF gene expression is controlled by a complex network of intersecting signalling pathways containing several dozen growth factors and cytokines, many of which induce VEGF expression in cancer cells. Future anti-angiogenic cancer therapies attempting to manipulate VEGF expression will have to take this complexity into account, as cancer cells may achieve VEGF upregulation both through HIF-related mechanisms as well as hypoxia-independent pathways, which are normally used to control VEGF expression dtuing development. The hardwired mechanisms of VEGF regulation are poorly understood and pose a major challenge for future research.

References 1. Michaeison IC. Retinal circulation in man and animals. Springfield, IL: Charles C. Thomas, 1954. 2. Shweiki D, Itin A, SofFer D et al. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992; 359(6398):843-845. 3. Leung DW, Cachianes G, Kuang WJ et al. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989; 246(4935): 1306-1309. 4. Folkman J. Tumor angiogenesis: Therapeutic implications. N Engl J Med 1971; 285(21):1182-1186. 5. Ferrara N. VEGF as a therapeutic target in cancer. Oncology 2005; 69(Suppl 3): 11-16. 6. Carmeliet P, Ferreira V, Breier G et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996; 380(6573):435-439. 7. Ferrara N, Carver-Moore K, Chen H et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 1996; 380(6573):439-442. 8. Forsythe JA, Jiang BH, Iyer NV et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 1996; 16(9):4604-4613. 9. Madan A, Curtin PT. A 24-base-pair sequence 3' to the human erythropoietin gene contains a hypoxia-responsive transcriptional enhancer. Proc Natl Acad Sci USA 1993; 90(9):3928-3932. 10. Wang GL, Semenza GL. General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia. Proc Natl Acad Sci USA 1993; 90(9):4304-4308. 11. Wang GL, Jiang BH, Rue EA et al. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular 0 2 tension. Proc Nad Acad Sci USA 1995; 92(12):5510-55l4. 12. Oosthuyse B, Moons L, Storkebaum E et al. Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Nat Genet 2001; 28(2):131-138. 13. Marti HH, Risau W. Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factor and its receptors. Proc Natl Acad Sci USA 1998; 95(26):15809-15814. 14. Levy AP. Hypoxic regulation of VEGF mRNA stabiHty by RNA-binding proteins. Trends Cardiovasc Med 1998; 8(6):246-250. 15. HoUams EM, Giles KM, Thomson AM et al. MRNA stabiHty and the control of gene expression: Implications for human disease. Neurochem Res 2002; 27(10):957-980.

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16. Meinsma D, Scheper W, Holthuizen PE et al. Site-specific cleavage of IGF-II mRNAs requires sequence elements from two distinct regions of the IGF-II gene. Nucleic Acids Res 1992; 20(19):5003-5009. 17. ClafFey KP, Shih SC, Mullen A et al. Identification of a human VPFATEGF 3' untranslated region mediating hypoxia-induced mRNA stability. Mol Biol Cell 1998; 9(2):469-481. 18. Zhao Z, Chang FC, Furneaux HM. The identification of an endonuclease that cleaves within an HuR binding site in mRNA. Nucleic Acids Res 2000; 28(14):2695-2701. 19. Nabors LB, Gillespie GY, Harkins L et al. HuR, a RNA stability factor, is expressed in malignant brain tumors and binds to adenine- and uridine-rich elements within the 3' untranslated regions of cytokine and angiogenic factor mRNAs. Cancer Res 2001; 61 (5):2154-2161. 20. Onesto C, Berra E, Grepin R et al. Poly(A)-binding protein-interacting protein 2, a strong regulator of vascular endothelial growth factor mRNA. J Biol Chem 2004; 279(33):34217-34226. 21. Ciais D, Cherradi N, Bailly S et al. Destabilization of vascular endothelial growth factor mRNA by the zinc-finger protein TISllb. Oncogene 2004; 23(53):8673-8680. 22. Gnarra JR, Zhou S, Merrill MJ et al. Post-transcriptional regulation of vascular endothelial growth factor mRNA by the product of the VHL tumor suppressor gene. Proc Natl Acad Sci USA 1996; 93(20):10589-10594. 23. Iliopoulos O, Levy AP, Jiang C et al. Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein. Proc Nad Acad Sci USA 1996; 93(20):10595-10599. 24. Laroia G, Sarkar B, Schneider RJ. Ubiquitin-dependent mechanism regulates rapid turnover of AU-rich cytokine mRNAs. Proc Nad Acad Sci USA 2002; 99 (4): 1842-1846. 25. Datta K, Mondal S, Sinha S et al. Role of elongin-binding domain of von Hippel Lindau gene product on HuR-mediated VPFA^EGF mRNA stability in renal cell carcinoma. Oncogene 2005; 24(53):7850-7858. 26. Shih SC, Mullen A, Abrams K et al. Role of protein kinase C isoforms in phorbol ester-induced vascular endothelial growth factor expression in human glioblastoma cells. J Biol Chem 1999; 274(22):15407-15414. 27. Pages G, Berra E, Milanini J et al. Stress-activated protein kinases (JNK and p38/HOG) are essential for vascular endothelial growth factor mRNA stabiUty. J Biol Chem 2000; 275(34):26484-26491. 28. Pain VM. Initiation of protein synthesis in eukaryotic cells. Eur J Biochem 1996; 236(3):747-771. 29. Kozak M. Pushing the limits of the scanning mechanism for initiation of translation. Gene 2002; 299(l-2):l-34. 30. Stein I, Itin A, Einat P et al. Translation of vascular endothelial growth factor mRNA by internal ribosome entry: Implications for translation under hypoxia. Mol Cell Biol 1998; 18(6):3112-3119. 31. Semenza G. Signal transduction to hypoxia-inducible factor 1. Biochem Pharmacol 2002; 64(5-6):993-998. 32. Xie K, Wei D, Shi Q et al. Constitutive and inducible expression and regulation of vascular endothelial growth factor. Cytokine Growth Factor Rev 2004; 15(5):297-324. 33. Milanini J, Vinals F, Pouyssegur J et al. p42/p44 MAP kinase module plays a key role in the transcriptional regulation of the vascular endothelial growth factor gene in fibroblasts. J Biol Chem 1998; 273(29):18165-18172. 34. Berra E, Milanini J, Richard DE et al. Signaling angiogenesis via p42/p44 MAP kinase and hypoxia. Biochem Pharmacol 2000; 60(8):1171-1178. 35. Pages G, Pouyssegur J. Transcriptional regulation of the Vascular Endothelial Growth Factor gene— a concert of activating factors. Cardiovasc Res 2005; 65(3):564-573. 36. Ryuto M, Ono M, Izumi H et al. Induction of vascular endothelial growth factor by tumor necrosis factor alpha in human glioma cells: Possible roles of SP-1. J Biol Chem 1996; 271(45):28220-28228. 37. Shi Q, Le X, Abbruzzese JL et al. Constitutive Spl activity is essential for differential constitutive expression of vascular endothelial growth factor in human pancreatic adenocarcinoma. Cancer Res 2001; 61(10):4143-4154. 38. Mukhopadhyay D, Knebelmann B, Cohen HT et al. The von Hippel-Lindau tumor suppressor gene product interacts with Spl to repress vascular endothelial growth factor promoter activity. Mol Cell Biol 1997; 17(9):5629-5639. 39. Salimath B, Marme D, Finkenzeller G. Expression of the vascular endothelial growth factor gene is inhibited by p73. Oncogene 2000; 19(31):3470-3476. 40. Niu G, Wright KL, Huang M et al. Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis. Oncogene 2002; 21(13):2000-2008. 41. Wei D, Le X, Zheng L et al. Stat3 activation regulates the expression of vascular endothelial growth factor and human pancreatic cancer angiogenesis and metastasis. Oncogene 2003; 22(3):319-329. 42. Michiels C, Minet E, Michel G et al. HIF-1 and AP-1 cooperate to increase gene expression in hypoxia: Role of MAP kinases. lUBMB Life 2001; 52(l-2):49-53.

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43. Gerald D, Berra E, Frapart YM et al. JunD reduces tumor angiogenesis by protecting cells from oxidative stress. Cell 2004; 118(6):781-794. AA, Gerhardt H, Golding M, Fruttiger M et al. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 2003; 161(6):1163-1177. 45. West H, Richardson WD, Fruttiger M. Stabilization of the retinal vascular network by reciprocal feedback between blood vessels and astrocytes. Development 2005; 132(8):1855-1862. 46. Vinores SA, Xiao WH, Aslam S et al. Implication of the hypoxia response element of the Vegf promoter in mouse models of retinal and choroidal neovascularization, but not retinal vascular development. J Cell Physiol 2006; 206(3):749-758. 47. Chen EY, Fujinaga M, Giaccia AJ. Hypoxic microenvironment within an embryo induces apoptosis and is essential for proper morphological development. Teratology 1999; 60(4):215'225. 48. Lee YM, Jeong CH, Koo SY et al. Determination of hypoxic region by hypoxia marker in developing mouse embryos in vivo: A possible signal for vessel development. Dev Dyn 2001; 220(2):175-186. 49. Lawson ND, Vogel AM, Weinstein BM. Sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation. Dev Cell 2002; 3(1):127-136. 50. Schwarz Q, Gu C, Fujisawa H et al. Vascular endothelial growth factor controls neuronal migration and cooperates with Sema3A to pattern distinct compartments of the facial nerve. Genes Dev 2004; 18(22):2822-2834. 51. Rosenstein JM, Krum JM. New roles for VEGF in nervous tissue—beyond blood vessels. Exp Neurol 2004; 187(2):246-253. 52. Storkebaum E, Lambrechts D, Carmeliet P. VEGF: Once regarded as a specific angiogenic factor, now implicated in neuroprotection. Bioessays 2004; 26(9):943-954. 53. Loureiro RM, D'Amore PA. Transcriptional regulation of vascular endothelial growth factor in cancer. Cytokine Grovirth Factor Rev 2005; 16(l):77-89.

CHAPTER 4

Embiyonic Vasculogenesis and Hematopoietic Specification Lauren C. Goldie, Melissa K. Nix and Karen K. Hirschi* Abstract

V

asculogenesis is the process by which blood vessels are formed de novo. In mammals, vasculogenesis occurs in parallel widi hematopoiesis, die formation of blood cells. Thus, it is debated whether vascular endothelial cells and blood cells are derived from a common progenitor. Whether or not this is the case, there certainly is commonality among regulatory factors that control the differentiation and differentiated function of both cell lineages. VEGF is a major regulator of both cell types and plays a critical role, in coordination with other signaling pathways and transcriptional regulators, in controlling the differentiation and behavior of endothelial and blood cells during early embryonic development, as further discussed herein.

Key Messages • • • • •

Blood cells and blood vessel endothelium develop in parallel, but it is still debated whether they develop from a shared precursor. VEGF signaling is critical for vascular plexus formation and hematopoiesis. VEGF signaling cooperates with other signaling pathways to promote endothelial cell specification, vascular plexus remodeling and hematopoetic specification. VEGF signaling is fine tuned via a collection of receptors and co-receptors to modulate endothelial cell behavior. The precise contribution of different VEGF signalii^ pathways to the r^julation of vascular development and hematopoiesis is not yet fixlly understood and needs to be researched further.

Introduction Vasculogenesis is the process by which blood vessels are formed de novo. This process first occurs in the embryonic yolk sac of mammalian embryos, and then later during development in the embryo proper. During gastrulation, embryonic ectodermal (epiblast) cells are recruited to the primitive streak where they undergo an epithelial to mesenchymal transition. These cells then migrate between the visceral endoderm and epiblast to form either mesoderm or definitive endoderm. In the yolk sac, the visceral endoderm is thought to elicit soluble signals which target the underlying mesoderm to induce the formation of primitive endothelial and hematopoietic cells, the first differentiated cell types to be produced in the mammalian embryo (Fig. 1). Primitive endothelial and hematopoietic cells coalesce to form blood islands that then fuse *Correspondlng Author: Karen K. Hirschi—Department of Pediatrics, Children's Nutrition Research Center and Department of Molecular and Cellular Biology, Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas 77030, USA. Email: [email protected]

VEGF in Development^ edited by Christiana Ruhrberg. ©2008 Landes Bioscience and Springer Science+Business Media.

Embryonic Vasculogenesis and Hematopoietic Specification

41

Figure 1. Molecular regulation of primitive hematopoietic specification and vascular remodeling during murine development. A) Schematic representation of the developing murine embryo, highlighting the primary sites of embryonic hematopoiesis. B) Schematic representation of endoderm-derived molecular signals with knov^n roles in the specification of mesodermal precursors. C) Schematic representation of primitive hematopoietic development within the yolk sac vascular plexus.

to form a primitive network of tubules known as a capillary or vascular plexus. Remodeling and maturation of the capillary plexus into a circulatory network requires the subsequent recruitment of mural cells (smooth muscle cells and pericytes) to form the outer blood vessel wall. Concurrent with vascular remodeling is the induction of definitive hematopoiesis, the formation of mature circulating blood cells. This chapter focuses on understanding the sequential processes of vasculogenesis and hematopoiesis, and the major signaling pathways that regulate these processes; a key regulatory factor is vascular endothelial growth factor (VEGF), the main focus of this text.

Endothelial Specification and Vasculogenesis During blood vessel development, endothelial cells are predominantly derived from mesoderm, as are blood cells. Mural cells are also largely derived from mesoderm, although neural crest and proepicardial organ cells also contribute mural cells to developing vessels. Vascular development has been well studied in several embryonic model systems including mouse, chick, quail, frog, and zebrafish, but perhaps best genetically manipulated in the murine system. Thus, our discussion of the molecular regulation of vasculogenesis and hematopoiesis will be based largely on insights gained from the study of the developing mouse; relevant insights from other model systems will also be discussed.

Formation of Mesodermal Precursors Murine blood vessel formation initiates during gastrulation, wherein posterior epiblast cells migrate through the primitive streak to form mesoderm,^ from which vascular and blood cells are differentiated. Several signals cooperate to promote mesoderm specification for blood vessel formation (Fig. IB). One signal needed for specification of mesoderm is the soluble factor bone morphogenic protein 4 (BMP4), whose expression prior to gastrulation is localized to the primitive streak. In the absence of BMP4, mesoderm fails to develop and thus mutant embryos arrest at the egg chamber stage."^ Another factor required for mesoderm formation is fibroblast growth factor 2 (FGF2, previously known as basic FGF or bFGF). Knockout inactivation of

42

VEGF in Development

FGF receptor 1 (FGFRl) results in an accumulation of epiblast cells, as they fail to migrate through the primitive streak and form mesoderm, further emphasizing the importance of FGF signaling in mesoderm formation.^ VEGF has critical roles in the subsequent stages of mesodermal commitment and differentiation, but unlike BMP4 and FGF2, VEGF is not known to play a role in mesoderm formation.

Commitment

of Mesodermal Precursors to the Endothelial

Lineage

During embryogenesis, commitment of multipotent mesodermal cells to an endothelial cell lineage is thought to be regulated by soluble signals derived from adjacent endodermal cells. In the murine yolk sac, visceral endoderm elicits signals needed for vascular induction, although the hierarchy of such signals is not yet clear. One such soluble effector is indian hedgehog (IHH) (Fig. IB). Using mouse embryo explant cultures lacking endoderm, it was discovered that IHH can respecify anterior epiblast cells, which normally form neuroectoderm, to form endothelial and hematopoietic cells instead, thus demonstrating sufficiency of IHH for vascular induction. IHH's downstream effector in this pathway may be BMP4, as it is up regulated in response to IHH signaling (Fig. IB). In support of this theory, aberrant vasculogenesis and hematopoiesis has been demonstrated in BMP4-null mutant mouse embryos that survive past the egg cylinder chamber stage.'^

Role of VEGFIKDR Signaling in Mesodermal of the Endothelial Lineage

Precursors

Synergistic signaling between IHH and endoderm-derived FGF2 is thought to promote the expression of the VEGF receptor KDR (also known as FLKl or VEGFR2) in adjacent mesodermal cells (Fig. IB). A role for FGF2 in the formation of endothelial precursors, angioblasts, from mesoderm has been demonstrated in the quail embryo, where explant cultures consisting of only mesoderm lack the ability to undergo angioblast formation unless treated with FGF2. Furthermore, isolated FGF2-treated epiblast cells are capable of forming endothelial cells in vitro. Perhaps the critical role of FGF2 in vivo during vascular induction is the up regulation of KDR. Once KDR is upregulated in mesodermal progenitors (not all of which will become endothelial cells'^), they become capable of responding to VEGF, which is initially produced only in the visceral endoderm of the mouse yolk sac.^ KDR-expressing mesodermal progenitors are exquisitely sensitive to the bioactive levels of VEGF, which therefore exerts a dose-dependent effect on vasculogenesis. This is evidenced by the fact that mice heterozygous for a null mutation in the Vegfa gene die in utero due to failed vascular development: In these mutants, endothelial cells differentiate, but in a delayed fashion, and this leads to disorganized blood vessels and disrupts hematopoietic cell formation. Similarly, embryos lacking KDR exhibit arrested vascular development and embryonic lethality. These mutants form endothelial precursors, but they fail to differentiate into mature endothelial cells, thereby halting blood island development and vascular plexus formation. ^^ The data obtained from the KDR null mice have been corroborated in both the avian and Xenopus systems. In quail embryos, KDR expression was knocked down and revealed that primitive endothelial cells developed in the absence of KDR, but no mature endothelial networks formed. ^^ In addition, it was demonstrated that quail mesoderm isolated from embryos can form hemangioblasts upon treatment with VEGF, even in the absence of contact with endoderm.^^ When VEGF was ectopically expressed in Xenopus embryos, large disorganized vascular structures containing mature endothelial cells formed. Taken together, these data indicate that VEGF is critically important for the differentiation and/or survival of mature endothelial cells.

Role ofVEGF/Neuropilin

Signaling in the Endothelial

Lineage

Recently, two novel VEGF receptors were discovered and termed neuropilin 1 and 2 (NRPl and NRP2). Originally, the neuropilins were identified in the nervous system as transmembrane receptors for class III semaphorins, which provide repulsive cues during the axon guidance of

Embryonic Vasculogenesis and Hematopoietic Specification

43

several embiyonic nerves.^ ' ^ Both NRPl and NRP2 are also expressed on endodielial cells, where diey function as isoform-specific VEGF receptors.^^'^'^ NRPl null mice are embryonic lethal between El2.5 and El3.5 due to cardiovascular defects, including defective aortic arch remodeling and formation of abnormal capillary networks in the brain and spinal cord.^^ In contrast, NRP2 null mice have been reported to be viable and fertile. ^^ However, ablation of both NRPl and NRP2 results in embryonic lethality by E8.5 in the mouse, as no organized blood vessels are formed, similar to the KDR and VEGF null phenotypes.^^ This demonstrates, once again, the necessity of precise VEGF signaling for early vascular development.

Capillary Plexus Formation As endothelial cells are committed and differentiated during vasculogenesis, their proliferation, migration and coalescence into a primitive capillary plexus must be well coordinated. VEGF signaling is critically important in this process, as it promotes endothelial cell proliferation^^'^^ and modulates migration."^"^'^^ FGF2 is also involved in the stimulation of endothelial cell proliferation,^^ and has been shown to function synergistically with VEGF. '^^'^^ The mitogenic effects of VEGF are thought to be mediated largely by the KDR receptor.^^'^^ In contrast, an alternative VEGF receptor termed FLTl (also known as VEGFRl) is thought to modulate VEGF s proliferative effects by sequestering locally available soluble VEGF. Consistent with this idea is the phenotype of embryos lacking FLTl: These mutants exhibit excessive endothelial cell formation, which leads to the formation of a disorganized vascular plexus incapable of remodeling into a functional circulatory network.^^'^^ In addition, a soluble form of FLTl termed sFLTl is thought to be critical to shape VEGF gradients in the environment of growing blood vessels to promote directional vessel growth.^^ These studies emphasize the fact that although VEGF's mitogenic effects are essential for capillary plexus formation, they must be balanced to prevent excessive endothelial cell proliferation and promote sprouting.

Vascular Remodeling Once a primitive endothelium has been formed and patterned, the next aspect of blood vessel development is remodeling of the plexus to promote the establishment of a mature circulatory network.

Endothelial Cell Proliferation, Migration and Survival Vascular remodeling is a complex process, in which a balance between signals to induce and inhibit endothelial cell proliferation must be reached. This process involves multiple signaling cascades as well as cell-cell and cell-matrix communications. Factors involved in maintaining the appropriate rate of endothelial cell proliferation include VEGF, FGF2, retinoic acid (RA), and transforming growth factor beta (TGFBl). VEGF and FGF2 are needed for the induction of endothelial cell proliferation. However counteractive anti-proliferative signals such as RA and TGFBl^^ are an equally essential requirement for appropriate blood vessel formation. Retinaldehyde dehydrogenase 2 (RALDH2) converts retinol (vitamin A) into its biologically active metabolite retinoic acid. Like other mesoderm modulating factors in the developing murine yolk sac, RALDH2 is expressed by the visceral endoderm, where active production of retinoic acid occurs. Retinoic acid is actively secreted from the visceral endoderm and interacts with retinoic acid receptors (RARAl/2) expressed by the vascular endothelium.^^ RARAl/ 2 signaling direcdy inhibits endothelial cell cycle progression via upregulation of two cell cycle inhibitors, the cyclin-dependent kinase inhibitors p21 (CDKNIA) and p27 ( C D K N I B ) , ^ and indirecdy suppresses growth via upregulation of the anti-proliferative cytokine TGFBl. TGFBl likely acts through the SMAD5 pathway to upregulate the production of extracellular matrix protein fibronectin, which then functions to promote visceral endoderm survival and signals via integrins present on vascular endothelium. Of particular importance are integrins alpha 5 beta 1 and alpha V beta 3, which both bind fibronectin, but eUcit opposite responses. Signalling of integrin alpha 5 beta 1 via a currently undefined intracellular signaling pathway

44

VEGF in Development

serves to block endothelial cell proliferation; in contrast, integrin alpha v beta 3 signals via the KDR/MAPK pathway to promote endotheUal cell proliferation.^^ The maintenance of a delicate equilibrium between these signaling events is necessary to regulate the proper branching and remodeling required for development of a mature vascular network.

Role ofMechanotransduction and Flow in the Maintenance of Blood Vessels Once blood vessels have formed, and likely concurrent with the remodeling process, fortification of the vessel wall is initiated in response to hemodynamic forces, which are exerted upon endothelial cells by the newly established circulatory flow. Genomic studies in vitro have identified a multitude of genes that are differentially regtdated by various types of hemodynamic forces, including laminar shear stress, turbulent shear stress, and disturbed flow. ' ^ These studies provide strong evidence that endothelial cells not only have the ability to "sense" hemodynamic forces, but that they are also capable of discriminating between different types of biomechanical stimuli. In partictdar, shear stress has been shown to promote endothelial cell survival via two main events, cell growth and inhibition of cell death (apoptosis). While the role of VEGF in endothelial response to shear stress is currendy unknown, its receptor KDR appears of great importance: KDR is a mechanosensor that converts mechanical stimuli into chemical signals, as both its expression and activation level increase in response to laminar shear stress. ^ Upon exposure to fluid shear stress, endothelial cells also upregulate KDR expression to activate bodi ERK and JNK kinases, which are downstream targets of the MAPK pathway. ^ Once these pathways are initiated, the transcription of immediate early response genes such as monocyte chemotactic protein 1 and FOS is upregulated to promote endothelial cell growth. Anti-apoptotic signaling pathways are also regulated by shear stress. Activation of the receptor tyrosine kinases KDR and TIE2 initiates a signaling cascade, in which activation of PI3K promotes the phosphorylation of AKT, which in turn triggers the upregulation of nitric oxide production and thereby elicits an antiapoptotic signal to endothelial cells in the blood vessel wall. ^ Endothelial expression of p21 drastically increases in response to increased nitric oxide production, resulting in the inhibition of endothelial cell apoptosis. Vice versa, loss of p21 significandy increases endothelial cell death in response to shear stress, demonstrating that p21 is one of the major factors mediating the anti-apoptotic effect of shear stress. In addition to its anti-apoptotic role, p21 creates a G l to S phase block via cyclin-dependent kinase phosphorylation of the retinoblastoma protein, ^ thus decreasing the rate of DNA synthesis during exposure to laminar shear stress.

Hemogenic Specification and Hematopoiesis Developmental Origin of Hematopoietic Progenitors In the mouse, primitive hematopoiesis is initiated in the yolk sac between embryonic day 7.0 and 7.5, producing predominandy nucleated erythroid cells expressing embryonic globin. A second wave of hematopoiesis is initiated in the yolk sac^^ and embryo proper between ElO.O and El 1.0,^^ when definitive hematopoietic stem cells (HSCs), capable of repopidating the neonate or adult blood system, arise in the aorta-gonad-mesonephros (AGM) region. Within the AGM, hematopoietic precursors are seen as clumps of cells that appear to bud from endothelial cells of the ventral wall of the dorsal aorta, and the umbilical and vitelline arteries. Thus, during definitive as well as primitive yolk sac hematopoiesis, there is a close spatial and temporal association between endothelial and hematopoietic cell development. Studies in both chick and mouse have shown that AGM-derived yolk sac cells, which give rise to endothelial cells of the yolk sac vasculature, also have the capacity to generate definitive hematopoietic cells in vitro. Together, these findings point to endothelium as the most likely source of definitive hematopoietic precursors in the developing embryo. However, the differing interpretation of the current body of experimental evidence has yielded three major theories for the origin and specification of the hematopoietic lineage (Fig. 2).

Embryonic Vasculogenesis and Hematopoietic Specification

45

Figure 2. Theories of hematopoietic specification during embryogenesis. Schematic representation of the three major alternative theories for the developmental emergence of primitive endothelial and hematopoietic lineages: (A) the bipotenfhemangioblast'', (B) "hemogenic" endothelium, and (C) gastrulation-specified endothelial and hematopoietic progenitors.

Endothelial and Hematopoietic Cells May Share a Common Progenitor In blood islands of the yolk sac, where the earliest hematopoietic cells appear, cells in the hematopoietic and endothelial lineage arise almost simultaneously from the extraembryonic paraxial mesoderm to form a primitive capillary plexus, in which primitive nucleated erythroblasts are intimately associated with maturing endothelial cells. This observation has led to the hypothesis that both lineages arise from a common precursor (Fig. 2A). This concept is supported by experimental evidence suggesting the shared expression of hematopoietic and endothelial-specific genes in both lineages. Even though the physical proximity of endothelial and hematopoietic cells in the developing embryo does not prove the existence of a common progenitor, supportive evidence for this concept is provided by in vitro studies, which demonstrate the formation of clonal blast colony-forming cells capable of generating both primitive hematopoietic and endothelial cell types under appropriate culture conditions. These cells express markers associated with both hematopoietic and vascular development, including j ^ j ^ 60-62 'jp^i 50,65 VE-cadherin^^ and GATAl .^^ Cell tracking studies of transgenic murine embryos with GFP targeted to the brachyury locus have also identified clonal progenitors in the gastrulating mouse embryo that are capable of forming blast cell colonies with ex vivo hematopoietic and vascular potential.^ Hematopoietic Cells May Be Derived from Specialized Hemogenic Endothelium An alternative theory, equally consistent with existing evidence, postulates that blood forming cells are specified from mesodermally-derived primitive endothelium known as hemogenic endothelium (Fig. 2B). Clusters of hematopoietic precursors that appear to derive directly from the vascular endothelium in vivo have been observed in developing blood vessels of numerous vertebrate species, and their appearance correlates with the developmental timing of definitive

46

VEGF in Development

hematopoietic cells. In the murine yolk sac and embryo proper, cells with blood-forming capacity have also been found to reside within the vascular endothelium. A population of cells termed side population cells has been isolated from murine yolk sac and embryo prior to the onset of circulation; this cell population resides within the vascular endothelium, expresses high levels of endothelial markers, and fails to generate blood cells in vitro; however, as development progresses, this cell population acquires hematopoietic potential and phenotypic characteristics similar to those of bone marrow side population cells, suggesting a developmental transition from an endothelial to a hematopoietic progenitor cell type. Culture studies also provided support for the model of hematopoietic specification from hemogenic endothelium. In particular, a population of primitive endothelial-like cells was isolated from human embryonic stem cell cultures, which expresses the endothelial markers PECAMl, KDRand VE-cadherin (PFV+), but not the hematopoetic lineage marker CD45; when treated with VEGF, these cells were shown to generate mature endothelial cells, whilst treatment with hematopoietic growth factors facilitated hematopoietic development. These studies therefore identified PFV+/CD45- cells as a distinct bipotent progenitor population capable of supporting the progressive development of the endothelial and hematopoietic lineages. Most significantly, these studies demonstrated the stepwise generation of hematopoietic and endothelial cell precursors from midtipotent embryonic cells, revealing a hierarchical organization of cell fate commitment, which supports the idea that hematopoietic and endothelial cells derive from a subset of embryonic endothelium with hemangioblastic properties. Endothelial and Hematopoietic Progenitors May Be Independently Specified during Gastnilation Other studies suggest that there is no common lineage between blood and endothelial cells, and that both are instead independently specified during gastrulation (Fig. 2C). Studies of the early primitive streak-stage embryo confirmed that cells derived from the primitive( streak preferentially contribute to the erythrocyte precursor population of the developing vascular blood islands.^ In contrast, cells derived from the primitive streak of mid-primitive streak-stage embrvos appear to contribute primarily to the endothelium, with little or no erythroid component. Perhaps most significantly, simultaneous contribution of primitive streak progenitors to blood islands and vitelline endothelium is rarely seen to occur at the same location within yolk sac mesoderm. These observations suggest that the allocation and recruitment of early progenitor cells to the endothelial and hematopoietic lineages likely occurs at different stages of gastrulation, and is therefore both spatially and temporally segregated during morphogenesis of the vitelline vessels. Sununary The relationship between hematopoietic precursors derived from hemogenic endothelium and putative yolk sac hemangioblasts remains unclear. Progenitors capable of reconstituting neonatal hematopoiesis arise in the yolk sac before circulation, and may be related to, or even derived from, the earlier born yolk sac hemangioblast. However, definitive hematopoietic stem cells that first arise in the AGM region are thought to be unrelated by lineage to yolk sac-derived precursors, because their development occurs independently of any circulation of cells from the yolk sac. Isolation of single cells that can give rise to both endotheUal and hematopoietic cells in vitro provides clear evidence of a common origin for the two lineages. However, it does not distinguish whether this common precursor makes a simple choice between the two lineages, or whether endothelium is formed first and blood forming cells are specified subsequendy, or whether there is a more primitive midtipotent precursor, whose potency remains undefined.

Developmental

Regulation of Hematopoietic

Progenitors

Although the cellular origin of hematopoietic cells is still debated, a number of signaling molecules have been shown to play critical roles in the specification and differentiation of blood cells.

Embryonic Vasculogenesis and Hematopoietic Specification

47

VEGF/KDR As previously discussed, KDR plays a key role in die developmental r^;ulation of hematopoiesis as well as vasculogenesis. Generally recognized as die earliest antigenic marker of developing endothelial cells, Kdr expression can be detected in mesodermally derived blood island progenitors as early as E7.0. ' Mice deficient in KDR fail to develop definitive hematopoietic cells capable of long-term engrafiment in an irradiated recipient, nor do thev makefimctionalblood vessels, and they therefore die in midgestation, between E8.5 and 9.5.^^' In chimeric a ^ r ^ t i o n smdies using wild-type mouse embryos, KDR null ES cells fail to contribute to primitive or definitive hematopoiesis in vivo, su^esting a cell autonomous requirement for KDR signaling in hematopoietic development.'^^ Furthermore, VEGF signalingfi-omthe yolk sac endoderm via KDR is required for the hematopoietic differentiation of mesoderm.^'^^''^^ Moreover, both vasculogenesis and hematopoiesis are impaired in mice homozygous for a hypomorphic VEGF allele, and decreased KDR activity.^ Although the findings described above demonstrate that VEGF/KDR signalling plays a crucial role in promoting hematopoiesis, it may not be essential for the initial specification of hematopoietic precursors KDR-deficient ES cells retain some hematopoietic potential in vitro^^ and KDR null mouse because embryos contain normal numbers of hematopoietic progenitors at E7.5, even though they are profoundly deficient at E8.5.^^ Rather than being required for specification, VEGF/KDR signalling may provide an important signal for the subsequent survival, migration and clonal expansion of hematopoeic progenitors. Consistent with this idea, KDR+ cells of mouse embryos carrying a hypomorphic Ve^a allele reach the yolk sac on time by E8.5, but are severely compromised in their ability to generate primitive erythroid precursors, perhaps because VEGF normally prolongs the life span of primitive erythroid progenitors by inhibiting apoptosis.'^ TALI The transcription factor TALI (also known as SCL) is first coexpressed with KDR around E7.0 in the visceral mesoderm and in vitro promotes the early stages of differentiation of mesoderm into cells of the hematopoietic lineage.^^' Gene targeting and chimera analyses demonstrated a requirement of TALI for the generation of both primitive and definitive hematopoietic lineages and for the appropriate remodeling of the yolk sac vasculature in vivo. Specifically, TALI null mutant embryos are embryonic lethal, as they fail to initiate yolk sac hematopoiesis. KDR+ cells isolated from differentiating TALI null ES cells also fail to generate either blood or endothelial cells in vitro,^^ suggesting that KDR signaling and TALI expression synergise during early hematopoietic development. GATAl, GATA2, L M 0 2 and RUNXl The transcription factors GATAl, GATA2, L M 0 2 and RU>DC1 also play important roles in the fate determination of the hematopoietic lineage. GATAl and GATA2 are essential for both embryonic and adult erythropoiesis: Loss of GATAl in knockout mouse embryos halts erythroid differentiation at the proerythroblast stage,^^ whilst loss of GATA2 function causes early embryonic lethality due to a primary defect in primitive hematopoiesis.^^ L M 0 2 is required for both vascular development and yolk sac hematopoiesis, and L M 0 2 null ES cells fail to contribute to the vascular endothelium of chimeric animals. ' Loss of function of any one of these three transcription factors yields a phenotype similar to that of TALI null mutant mice, consistent with the idea that L M 0 2 may function cooperatively with GATAl and TALI to promote the specification of erythroid cells. ^^ Definitive, but not primitive, hematopoiesis is also dependent on the transcription factor RU>DC1. RUNXl null mutant embryos undergo primitive yolk sac hematopoiesis normally, but die between El 1.0 and El2.0 due to a failure of definitive hematopoiesis. Z^cZ knock-in mice revealed expression oi Runxl in a subpopulation of cells in the yolk sac endothelium and in the floor of the dorsal aorta, suggesting that RUlSDCl supports the differentiation of hemogenic endothelium in vivo, which contributes to definitive hematopoiesis. Consistent with this idea, aortic clusters of hematopoietic cells are absent in RU>DC1 null mutants,^^ and in vitro RU>DC1 null embryoid bodies produce clonal blast colony-forming cells at reduced numbers.

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VEGF in Development

Conclusions and Future Directions Experimentation in several embryonic model systems by many different laboratories has revealed that VEGF signaling is critical for the formation of an initial vascular plexus from multipotent mesodermal progenitors. Continued VEGF signaling, fine-tuned via its receptors and coreceptors, then modulates endothelial cell behavior to establish a functional circulatory network. Other signaling pathways function coordinately with VEGF to mediate vascular plexus remodeling, endothelial cell specification and mural cell recruitment. The formation of blood vessels and blood cells occurs concomitandy. The VEGF receptor KDR is widely recognized as a common marker of all cells possessing putative hemangioblast properties. Cells that retain KDR activity have endothelial potential, whereas cells that activate hematopoietic transcription factors such as TALI and RUNXl gain hematopoietic activity. A subset of KDR-expressing cells that are TAL1+ may represent primitive hemangioblasts capable of initiating primitive yolk sac erythropoiesis, while KDR-expressing cells that are both TAL+ and RUNXl + represent definitive precursors for definitive hematopoiesis in the AGM and yolk sac.^^ This model of successive diversion of subsets of VEGF-responsive, KDR-expressing cells perhaps best explains our current understanding of the formation of endothelial, primitive hematopoietic and definitive hematopoietic precursors in the early embryo. However, continued research is needed to fully understand the complexity of VEGF signaling. Moreover, we need to increase our efforts to elucidate how VEGF signaling cooperates with other signaling pathways during vascular development and hematopoiesis. Insights gained from ongoing work in developmental model systems will likely benefit the optimization of clinical therapies for those prevalent diseases that are associated with disrupted blood cell and blood vessel formation.

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69. Dumont DJ, Fong GH, Puri MC et al. Vascularization of the mouse embryo: a study of Flk-1, Tek, Tie and vascular endothelial growth factor expression during development. Dev Dyn 1995; 203:80-92. 70. Shalaby F, Ho J, Stanford WL et al. A requirement for Flkl in primitive and definitive hematopoiesis and vasculogenesis. Cell 1997; 89:981-990. 71. Martin R, Lahlil R, Damert A et al. SCL interacts with VEGF to suppress apoptosis at the onset of hematopoiesis. Development 2003; 131:693-702. 72. Schuh AC, Faloon P, Hu Q-L et al. In vitro hematopoietic and endothelial potential of flk-1-/embryonic stem cells and embryos. Proc Nad Acad Sci U S A 1999; 96:2159-2164. 73. Elefanty AG, Begley CG, Hartley L et al. SCL expression in the mouse embryo detected with a targeted lacZ reporter gene demonstrates its localization to hematopoietic, vascular, and neural tissues. Blood 1999; 94:3754-3763. 74. Robertson SM, Kennedy M, Shannon JM, Keller G. A transitional stage in the commitment of mesoderm to hematopoiesis requiring the transcription factor SCL/Tal-1. Development 2000; 127:2447-2459. 75. Visvader JE, Fujiwara Y, Orkin SH. Unsuspected role for the T-cell leukemia protein SCL/Tal-1 in vascular development. Genes Dev 1998; 12(473-479). 7G. Shivdasani RA, Mayer EL, Orkin SH. Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein Tal-1/SCL. Nature 1995; 373:432-434. 77. Robb L, Begley CG. The helix-loop-helix gene SCL: implicated in T-cell acute lymphoblastic leukaemia and in normal haematopoietic development. Int J Biochem Cell Biol 1996; 28:609-618. 78. Ema M, Faloon P, Zhang WJ et al. Combinatorial effects of Flkl and Tall on vascular and hematopoietic development in the mouse. Genes Dev 2003; 17:380-393. 79. Fujiwara Y, Browne CP, Cunniff^K et al. Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1. Proc Natl Acad Sci U S A 1996; 93:12355-12358. 80. Tsai FY, Keller G, Kuo FC et al. An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature 1994; 371:221-226. 81. Yamada Y, Pannell R, Forster A, Rabbitts TH. The oncogenic LIM-only transcription factor Lmo2 regulates angiogenesis but not vasculogenesis in mice. Proc Natl Acad Sci U S A 2000; 97(l):320-324. 82. Warren AJ, CoUedge WH, Carlton MB et al. The oncogenic cysteine-rich LIM domain protein rbtn2 is essential for erythroid development. Cell 1994; 78:45-57. 83. Okuda T, van Deursen J, Hiebert SW et al. AMLl, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell 1996; 84:321-330. 84. North TE, de Bruijn MF, Stacy T et al. Runxl expression marks long-term repopulating hematopoietic stem cells in the midgestation mouse embryo. Immunity 2002; 16(5):661-672. 85. North T, Gu TL, Stacy T et al. Cbfa2 is required for the formation of intra-aortic hematopoietic clusters. Development 1999; 126:2563-2575. 86. Lacaud G, Gore L, Kennedy M et al. Runxl is essential for hematopoietic commitment at the hemangioblast st£^e of development in vitro. Blood 2002; 100:458-466. 87. Ema M, Rossant J. Cell fate decisions in early blood vessel formation. Trends Cardiovasc Med 2003; 13(6):254-259.

CHAPTER 5

Role of VEGF in Organogenesis Jody J. Haigh* Abstract

T

he cardiovascular system, consisting of the heart, blood vessels and hematopoietic cells, is the first organ system to develop in vertebrates and is essential for providing oxygen and nutrients to the embryo and adult organs. Work done predominandy using the mouse and zebrafish as model systems has demonstrated that Vascular Endothelial Growth Factor (VEGF, also known as VEGFA) and its receptors KDR (FLK1/VEGFR2), FLTl (VEGFRl), NRPl and NRP2 play essential roles in many different aspects of cardiovascular development, including endothelial cell differentiation, migration and survival as well as heart formation and hematopoiesis. This review will summarize the approaches taken and conclusions reached in dissecting the role ofVEGF signalling in vivo during the development of the early cardiovasculature and other organ systems. The VEGF-mediated assembly of a functional vasculature is also a prerequisite for the proper formation of other organs and for tissue homeostasis, because blood vessels deliver oxygen and nutrients and vascular endothelium provides inductive signals to other tissues. Particular emphasis will therefore be placed in this review on the cellidar interactions between vascular endothelium and developing organ systems, in addition to a discussion of the role of VEGF in modulating the behavior of nonendothelial cell populations.

Key Messages • • •

VEGF plays a central role in the development of the cardiovasculature. VEGF supports organogenesis indirecdy by promoting vascular development. VEGF supports organogenesis direcdy by acting on various non-endothelial cell types.

Introduction VEGF is initially expressed at high levels in the yolk sac and in embryonic sites of vessel formation to support the assembly of a cardiovascular system. However, in the embryo, VEGF continues to be expressed after the onset of blood vessel formation, consistent with the idea that it is instructive not only for cardiovascular development, but also for the development of other organ systems. In the adult, VEGF expression becomes restricted to specialized cell types in organs containing fenestrated endothelium, for example the kidney and pituitary. In addition, VEGF is up-regulated to mediate physiological angiogenesis during menstruation, ovidation and in wound healing. The expression of VEGF in adults is also induced by environmental stress caused by hypoxia, anemia, myocardial ischemia, and tumor progression to initiate neovascularisation.'^ Hypoxia up-regulates transcription of the gene encoding VEGF iye^d) by activating the hypoxia-inducible factors HIFIA and HIF2A (formerly known as HIFl alpha *Jody J. Haigh—Vascular Cell Biology Unit, Department for Molecular Biomedical Research (DMBR), Flanders Interuniversity Institute for Biotechnology (VIB)/Ghent University, Technologiepark 927, B9052 Ghent (Zwijnaarde), Belgium. Email: [email protected]

VEGF in Development edited by Christiana Ruhrberg. ©2008 Landes Bioscience and Springer Science+Business Media.

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and HIF2 alpha; see Chapter by 3 M. Fruttiger).^' Several major growth factors up-regulate VEGF, including epidermal growth factor (EGF), insidin-like growth factor (IGFl), fibroblast growth factor 2 (FGF2; also known as basic FGF), fibroblast growth factor 7 (FGF7; formerly known as keratinocyte growth factor, KGF), platelet-derived growth factor (PDGF) and the tumour growth factors TGFA and TGFB (formerly known as TGF alpha and beta).^ In addition, inflammatory cytokines and hormones have been shown to induce VEGF expression. '^ The mouse VEGF gene iVegfa) gives rise by alternative mRNA splicing and proteolytic processing to three major isoforms termed VEGF 120, VEGF 164 and VEGF 188 (see Chapter 1 by Y.-S. Ng).^ The corresponding human VEGF isoforms are one amino acid residue larger and therefore termed VEGF 121, VEGF 165 and VEGF189. In addition, a number of less abundant human isoforms have been described, including VEGF 145 VEGF 183, VEGF206. The VEGF isoforms differ not only in their molecular mass, but also in their solubility and receptor binding characteristics. VEGG188ArEGF189 and VEGF206 contain exons 6 and 7, which encode heparin-binding domains and mediate binding to heparan sulphate proteoglycan (HSPG) proteins in the extracellular matrix (ECM), and on the cell surface and ECM. VEGF 120/VEGF121 lacks the domains encoded by exons 6 and 7 and is therefore the most diffiisible isoform. VEGF 164A^EGF 165 contains the domain encoded by exon 7, but not exon 6, and accordingly has intermediate properties; 50-70% of this isoform remains associated with the cell surface and ECM. Due to the fact that these isoforms differ in their potential to bind to HSPGs, it is believed that they are differentially distributed in the environment of VEGF-secreting cells. The highest levels ofVEGF188 are detected in organs that are vascularized initially by vasculogenesis (e.g., lung, heart and liver), while organs vascularized primarily by angiogenesis, including brain, eye, muscle and kidney have higher levels of VEGF 164 and VEGF 120. The functional roles of these isoforms in organogenesis are described in more detail below.

Approaches to Study VEGF during Organogenesis Exon 3 is contained in all known VEGF isoforms. The targeted deletion of a single Vegfa allele by neomycin gene insertion into this exon leads to embryonic lethality due to abnormal blood vessel and heart development between E l l and E12, d e m o n s t r a t i n g haploinsufficiency.^^*^^ Using high G418 selection of the Vegfa+/- neomycin-resistant embryonic stem (ES) cells, Vegfa-/- ES cells could be produced, and these were used to make Vegfa-/ - embryos by tetraploid embryo-ES cell complementation approaches. The resultant Vegfa-/embryos were even more severely compromised than Vegfa+/- embryos and died between E9.5 and ElO owing to severe defects in vasculogenesis, angiogenesis and heart development, which was accompanied by tissue necrosis/apoptosis. Given the relatively early mid-gestational lethality associated with targeted VEGF gene inactivation, alternative strategies have been employed to examine the role of VEGF during organogenesis and in the adult: VEGF function has been ablated at the protein level with sequestering antibodies, small molecule inhibitors that interfere with receptor tyrosine kinase signaling, or a soluble truncated chimeric VEGF receptor termed mFLTl(l-3)-IgG, which consists of the FLTl extracellular domain fused to an IgG-Fc domain and sequesters VEGF protein with high affinity. Ubiquitous inducible ablation of the conditional VEGF allele or administration of mFLTl(l-3)-IgG causes lethality and severe multi-organ abnormalities in neonatal mice.^^ The defects in the lung, heart and kidney compromised the health status of these two types of mice severely, and the specific functions of VEGF in other organs were therefore difficult to deduce. To achieve a partial loss of VEGF function, different conditional Vegfa alleles containing paired LoxP sites have been created, which can be targeted with CRE recombinase to impair VEGF expression in a spatiotemporally defined manner {Cre/LoxP technology). Two different Cre/LoxP approaches have been used. Firsdy, mice in which exon 3 of VEGF is flanked by LoxP sites have been generated. ^^ In these mice, CRE-mediated excision deletes exon 3, which is present in all VEGF isoforms, and this results in loss ofVEGF production. ^^'^ ^ This conditional

54

VEGF in Development

Vegfa allele can be used to delete VEGF in a cell/tissue specific manner when mated to mouse strains expressing CRE recombinase under an appropriate promoter. An alternative approach was taken to understand the individual roles of the main VEGF isoforms (see Chapter 1 by Y.S. Ng). To create mice expressing only VEGF 120, exons 6 and 7 of the Vegfa gene were flanked by LoxP sites and deleted in ES cells by CRE-mediated excision. ^^ These targeted ES cells were then used to make mice expressing VEGF 120, but not VEGF 164 or VEGF 188. To create complementary mice expressing VEGF 164 or VEGF 188 only, a knock-in approach was used: cDNA sequences corresponding to exons 4,5,7,8 (in the case of VEGF 164) or 4-8 (in the case of VEGF 188) were inserted into the genomic Vegfii locus to remove intervening intron sequences and therefore abolish alternative splicing. Characterization of the phenotypical alterations present in the VEGF isoform-specific mice and the cell/tissue-specific VEGF mutants has contributed gready to our understanding of VEGF function in organogenesis and in adult life. Mice that express only the VEGF 120 isoform {Vegfiil20/120 mice) make normal overall VEGF levels, which permit the formation of sufficient numbers of endothelial cells and therefore vasculogenesis. Accordingly, these mice survive to birth. However, they do show defects in many organ systems, including the heart,^^'^^ retina,^^'^^'^^ bone,^^'^^ kidney^^ and lung.^ Most Vegfal20/ 120 mice die neonatally due to impaired cardiac fimction, with only 0.5% of animals living to postnatal day 12.^^ An important consideration in interpreting the defects of Vegfal20/120 mice is that they do not only lack VEGF 164 and VEGF 188, but also make an increased amount of VEGF 120 to compensate for the loss of the other two major splice forms. It has therefore been suggested that the relative balance of the different VEGF isoforms, rather than their absolute levels, is critical for angiogenic vessel patterning. About half of the Vegfal88/188 mice die prenatally due to severe defects in aortic arch remodeling, and the surviving half show defects in artery development in the eye and defects in epiphyseal bone vascularization.^ '^^'^^ In contrast to Vegfal 20/120 znd Vegfal 88/188 mice, mice that only express VEGF 164 {Vegfa 164/164) or compound Vegfal20/188 mice develop normally with no discernible phenotypes. ' The vascular phenotypes of VEGF isoform mutants are likely related to the differential localization of VEGF isoforms in the extracellular space, which provides a control point for regulating vascular branching morphogenesis. Microvessels from mouse embryos expressing only the VEGF 120 isoform have an abnormally large diameter and exhibit a decrease in branch formation, which is linked to a delocalization of secreted VEGF protein in the extracellular space. In contrast, mice expressing only the heparin-binding VEGF 188 isoform exhibit ectopic vessel branches, which appear long and thin. Intriguingly, the vessel branching and morphogenesis defects of Vegfal20/120 and Vegfal88/188 mice are rescued in compound mutant offspring of these mice that express both the VEGF 120 and VEGF 188 isoforms, even though they lack VEGF 164 (Vegftil20/188 mice). It therefore appears that the growing vasculature migrates along positional guidance cues provided by angiogenic VEGF gradients, which are normally composed of soluble and matrix-binding VEGF isoforms. This VEGF gradient can either be established by the coexpression of VEGF 120 and VEGF 188 or by VEGF 164 alone, as VEGF 164 is able to diffuse from the secreting cell, but also binds to and cooperatively signals with extracellular matrix molecules such as heparin sulfate proteoglycans. At the cellular level, VEGF gradients promote the formation of endothelial "tip-cells" at the front of growing vessels, which extend long filopodia presumably to seek out highest VEGF concentrations. ' Differential VEGF isoform expression does not only control developmental angiogenesis, but is likely to contribute also to physiological and pathological vessel growth in human adults. As an example, the different VEGF isoforms are differentially expressed in humans after submaximal exercise. ^^ Moreover, a novel splice variant of VEGF 165 termed VEGF 165b may provide a natural inhibitor of VEGFl65-induced angiogenesis, but is down-regulated in renal cell carcinoma, possibly supporting the switch from an anti-angiogenic to a pro-angiogenic phenotype during tumour development.^^ It has recentiy been demonstrated that VEGF 165, but not VEGF 121 specifically interacts with P-amyloid plaques in Alzheimer's disease and may prevent p-amyloid- induced formation of neurotoxic reactive oxygen species.'^^

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55

In order to increase our understanding of the individual roles of the three main VEGF isoforms, we have conditionally targeted each of these isoforms to the ubiquitously expressed Rosa26\oajiSy creating mice in which any one of the three isoforms can be selectively overexpressed in a cell type-specific fashion (Haigh et al, unpublished). Transgene expression is under the transcriptional control of the endogenous Rosa26^TomotcT, therefore Cr